Novel tumor necrosis factor receptor homologs and nucleic acids encoding the same

ABSTRACT

The present invention is directed to novel polypeptides having homology to members of the tumor necrosis factor receptor family and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

CROSS REFERENCE TO RELATED APPLICATION

This is a divisional application of U.S. patent application Ser. No.10/243,157 filed Sep. 12, 2002, which is a divisional application ofU.S. patent application Ser. No. 09/548,130 filed Apr. 12, 2000 (nowU.S. Pat. No. 6,534,061), which is a non-provisional applicationclaiming priority under Section 119(e) to U.S. Provisional PatentApplication Ser. No. 60/128,849 filed Apr. 12, 199, now abandoned, theentire disclosures of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of novel DNA and to the recombinant production of novelpolypeptides having homology to tumor necrosis factor receptors,designated herein as “DNA98853” polypeptides and “DNA101848”polypeptides.

BACKGROUND OF THE INVENTION

Control of cell numbers in mammals is believed to be determined, inpart, by a balance between cell proliferation and cell death. One formof cell death, sometimes referred to as necrotic cell death, istypically characterized as a pathologic form of cell death resultingfrom some trauma or cellular injury. In contrast, there is another,“physiologic” form of cell death which usually proceeds in an orderly orcontrolled manner. This orderly or controlled form of cell death isoften referred to as “apoptosis” [see, e.g., Barr et al.,Bio/Technology, 12:487-493 (1994); Steller et al., Science,267:1445-1449 (1995)]. Apoptotic cell death naturally occurs in manyphysiological processes, including embryonic development and clonalselection in the immune system [Itoh et al., Cell, 66:233-243 (1991)].Decreased levels of apoptotic cell death have been associated with avariety of pathological conditions, including cancer, lupus, and herpesvirus infection [Thompson, Science, 267:1456-1462 (1995)]. Increasedlevels of apoptotic cell death may be associated with a variety of otherpathological conditions, including AIDS, Alzheimer's disease,Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis,retinitis pigmentosa, cerebellar degeneration, aplastic anemia,myocardial infarction, stroke, reperfusion injury, and toxin-inducedliver disease [see, Thompson, supra].

Apoptotic cell death is typically accompanied by one or morecharacteristic morphological and biochemical changes in cells, such ascondensation of cytoplasm, loss of plasma membrane microvilli,segmentation of the nucleus, degradation of chromosomal DNA or loss ofmitochondrial function. A variety of extrinsic and intrinsic signals arebelieved to trigger or induce such morphological and biochemicalcellular changes [Raff, Nature, 356:397-400 (1992); Steller, supra;Sachs et al., Blood, 82:15 (1993)]. For instance, they can be triggeredby hormonal stimuli, such as glucocorticoid hormones for immaturethymocytes, as well as withdrawal of certain growth factors[Watanabe-Fukunaga et al., Nature, 356:314-317 (1992)]. Also, someidentified oncogenes such as myc, rel, and E1A, and tumor suppressors,like p53, have been reported to have a role in inducing apoptosis.Certain chemotherapy drugs and some forms of radiation have likewisebeen observed to have apoptosis-inducing activity [Thompson, supra].

Various molecules, such as tumor necrosis factor-α (“TNF-α”), tumornecrosis factor-β (“TNF-β” or “lymphotoxin-α”), lymphotoxin-β (“LT-β”),CD30 ligand, CD27 ligand, CD40 ligand, OX-40 ligand, 4-1BB ligand, Apo-1ligand (also referred to as Fas ligand or CD95 ligand), Apo-2 ligand(also referred to as TRAIL), Apo-3 ligand (also referred to as TWEAK),EDA and EDA-A2 have been identified as members of the tumor necrosisfactor (“TNF”) family of cytokines [See, e.g., Gruss and Dower, Blood,85:3378-3404 (1995); Pitti et al., J. Biol. Chem., 271:12687-12690(1996); Wiley et al., Immunity, 3:673-682 (1995); Browning et al., Cell72:847-856 (1993); Armitage et al. Nature, 357:80-82 (1992), WO 97/01633published Jan. 16, 1997; WO 97/25428 published Jul. 17, 1997; Marsterset al., Curr. Biol., 8:525-528 (1998); Chicheportiche et al., Biol.Chem., 272:32401-32410 (1997); Bayes et al., Human Molecular Genetics,7:1661-1669 (1998); Kere et al., Nature Genetics, 13:409-416 (1996)].Among these molecules, TNF-α, TNF-β, CD30 ligand, 4-1BB ligand, Apo-1ligand, Apo-2 ligand (TRAIL) and Apo-3 ligand (TWEAK) have been reportedto be involved in apoptotic cell death. Both TNF-α and TNF-β have beenreported to induce apoptotic death in susceptible tumor cells [Schmid etal., Proc. Natl. Acad. Sci., 83:1881 (1986); Dealtry et al., Eur. J.Immunol. 17:689 (1987)]. Zheng et al. have reported that TNF-α isinvolved in post-stimulation apoptosis of CD8-positive T cells [Zheng etal., Nature, 377:348-351 (1995)]. Other investigators have reported thatCD30 ligand may be involved in deletion of self-reactive T cells in thethymus [Amakawa et al., Cold Spring Harbor Laboratory Symposium onProgrammed Cell Death, Abstr. No. 10, (1995)].

Mutations in the mouse Fas/Apo-1 receptor or ligand genes (called lprand gld, respectively) have been associated with some autoimmunedisorders, indicating that Apo-1 ligand may play a role in regulatingthe clonal deletion of self-reactive lymphocytes in the periphery[Krammer et al., Curr. Op. Immunol., 6:279-289 (1994); Nagata et al.,Science, 267:1449-1456 (1995)]. Apo-1 ligand is also reported to inducepost-stimulation apoptosis in CD4-positive T lymphocytes and in Blymphocytes, and may be involved in the elimination of activatedlymphocytes when their function is no longer needed [Krammer et al.,supra; Nagata et al., supra]. Agonist mouse monoclonal antibodiesspecifically binding to the Apo-1 receptor have been reported to exhibitcell killing activity that is comparable to or similar to that of TNF-α[Yonehara et al., J. Exp. Med., 169:1747-1756 (1989)].

Induction of various cellular responses mediated by such TNF familycytokines is believed to be initiated by their binding to specific cellreceptors. Two distinct TNF receptors of approximately 55-kDa (TNFR1)and 75-kDa (TNFR2) have been identified [Hohman et al., J. Biol. Chem.,264:14927-14934 (1989); Brockhaus et al., Proc. Natl. Acad. Sci.,87:3127-3131 (1990); EP 417,563, published Mar. 20, 1991] and human andmouse cDNAs corresponding to both receptor types have been isolated andcharacterized [Loetscher et al., Cell, 61:351 (1990); Schall et al.,Cell, 61:361 (1990); Smith et al., Science, 248:1019-1023 (1990); Lewiset al., Proc. Natl. Acad. Sci., 88:2830-2834 (1991); Goodwin et al.,Mol. Cell. Biol., 11:3020-3026 (1991)]. Extensive polymorphisms havebeen associated with both TNF receptor genes [see, e.g., Takao et al.,Immunogenetics, 37:199-203 (1993)]. Both TNFRs share the typicalstructure of cell surface receptors including extracellular,transmembrane and intracellular regions. The extracellular portions ofboth receptors are found naturally also as soluble TNF-binding proteins[Nophar, Y. et al., EMBO J. 9:3269 (1990); and Kohno, T. et al., Proc.Natl. Acad. Sci. U.S.A., 87:8331 (1990)]. The cloning of recombinantsoluble TNF receptors was reported by Hale et al. [J. Cell. Biochem.Supplement 15F, 1991, p. 113 (P424)].

The extracellular portion of type 1 and type 2 TNFRs (TNFR1 and TNFR2)contains a repetitive amino acid sequence pattern of four cysteine-richdomains (CRDs) designated 1 through 4, starting from the NH₂-terminus.Each CRD is about 40 amino acids long and contains 4 to 6 cysteineresidues at positions which are well conserved [Schall et al., supra;Loetscher et al., supra; Smith et al., supra; Nophar et al., supra;Kohno et al., supra]. In TNFR1, the approximate boundaries of the fourCRDs are as follows: CRD1-amino acids 14 to about 53; CRD2-amino acidsfrom about 54 to about 97; CRD3-amino acids from about 98 to about 138;CRD4-amino acids from about 139 to about 167. In TNFR2, CRD1 includesamino acids 17 to about 54; CRD2-amino acids from about 55 to about 97;CRD3-amino acids from about 98 to about 140; and CRD4-amino acids fromabout 141 to about 179 [Banner et al., Cell, 73:431-435 (1993)]. Thepotential role of the CRDs in ligand binding is also described by Banneret al., supra.

A similar repetitive pattern of CRDs exists in several othercell-surface proteins, including the p75 nerve growth factor receptor(NGFR) [Johnson et al., Cell, 47:545 (1986); Radeke et al., Nature,325:593 (1987)], the B cell antigen CD40 [Stamenkovic et al., EMBO J.,8:1403 (1989)], the T cell antigen OX40 [Mallet et al., EMBO J., 9:1063(1990)] and the Fas antigen [Yonehara et al., supra and Itoh et al.,Cell 66:233-243 (1991)]. CRDs are also found in the soluble TNFR(sTNFR)-like T2 proteins of the Shope and myxoma poxviruses [Upton etal., Virology, 160:20-29 (1987); Smith et al., Biochem. Biophys. Res.Commun., 176:335 (1991); Upton et al., Virology, 184:370 (1991)].Optimal alignment of these sequences indicates that the positions of thecysteine residues are well conserved. These receptors are sometimescollectively referred to as members of the TNF/NGF receptor superfamily.Recent studies on p75NGFR showed that the deletion of CRD1 [Welcher, A.A. et al., Proc. Natl. Acad. Sci. USA, 88:159-163 (1991)] or a 5-aminoacid insertion in this domain [Yan, H. and Chao, M. V., J. Biol. Chem.,266:12099-12104 (1991)] had little or no effect on NGF binding [Yan, H.and Chao, M. V., supra]. p75 NGFR contains a proline-rich stretch ofabout 60 amino acids, between its CRD4 and transmembrane region, whichis not involved in NGF binding [Peetre, C. et al., Eur. J. Hematol.,41:414-419 (1988); Seckinger, P. et al., J. Biol. Chem., 264:11966-11973(1989); Yan, H. and Chao, M. V., supra]. A similar proline-rich regionis found in TNFR2 but not in TNFR1.

The TNF family ligands identified to date, with the exception oflymphotoxin-α, are type II transmembrane proteins, whose C-terminus isextracellular. In contrast, most receptors in the TNF receptor (TNFR)family identified to date are type I transmembrane proteins. In both theTNF ligand and receptor families, however, homology identified betweenfamily members has been found mainly in the extracellular domain(“ECD”). Several of the TNF family cytokines, including TNF-α, Apo-1ligand and CD40 ligand, are cleaved proteolytically at the cell surface;the resulting protein in each case typically forms a homotrimericmolecule that functions as a soluble cytokine. TNF receptor familyproteins are also usually cleaved proteolytically to release solublereceptor ECDs that can function as inhibitors of the cognate cytokines.

Recently, other members of the TNFR family have been identified. Suchnewly identified members of the TNFR family include CAR1, HVEM andosteoprotegerin (OPG) [Brojatsch et al., Cell 87:845-855 (1996);Montgomery et al., Cell 87:427-436 (1996); Marsters et al., J. Biol.Chem., 272:14029-14032 (1997); Simonet et al., Cell 89:309-319 (1997)].Unlike other known TNFR-like molecules, Simonet et al., supra, reportthat OPG contains no hydrophobic transmembrane-spanning sequence.

Another new member of the TNF/NGF receptor family has been identified inmouse, a receptor referred to as “GITR” for “glucocorticoid-inducedtumor necrosis factor receptor family-related gene” [Nocentini et al.,Proc. Natl. Acad. Sci. USA 94:6216-6221 (1997)]. The mouse GITR receptoris a 228 amino acid type I transmembrane protein that is expressed innormal mouse T lymphocytes from thymus, spleen and lymph nodes.Expression of the mouse GITR receptor was induced in T lymphocytes uponactivation with anti-CD3 antibodies, Con A or phorbol 12-myristate13-acetate.

In Marsters et al., Curr. Biol., 6:750 (1996), investigators describe afull length native sequence human polypeptide, called Apo-3, whichexhibits similarity to the TNFR family in its extracellularcysteine-rich repeats and resembles TNFR1 and CD95 in that it contains acytoplasmic death domain sequence [see also Marsters et al., Curr.Biol., 6:1669 (1996)]. Apo-3 has also been referred to by otherinvestigators as DR3, wsl-1, TRAMP, and LARD [Chinnaiyan et al.,Science, 274:990 (1996); Kitson et al., Nature, 384:372 (1996); Bodmeret al., Immunity, 6:79 (1997); Screaton et al., Proc. Natl. Acad. Sci.,94:4615-4619 (1997)].

Pan et al. have disclosed another TNF receptor family member referred toas “DR4” [Pan et al., Science, 276:111-113 (1997)]. The DR4 was reportedto contain a cytoplasmic death domain capable of engaging the cellsuicide apparatus. Pan et al. disclose that DR4 is believed to be areceptor for the ligand known as Apo-2 ligand or TRAIL.

In Sheridan et al., Science, 277:818-821 (1997) and Pan et al., Science,277:815-818 (1997), another molecule believed to be a receptor for theApo-2 ligand (TRAIL) is described. That molecule is referred to as DR5(it has also been alternatively referred to as Apo-2; TRAIL-R2, TRICK2or KILLER [Screaton et al., Curr. Biol., 7:693-696 (1997); Walczak etal., EMBO J., 16:5386-5387 (1997); Wu et al., Nature Genetics,17:141-143 (1997)]. Like DR4, DR5 is reported to contain a cytoplasmicdeath domain and be capable of signaling apoptosis.

Yet another death domain-containing receptor, DR6, was recentlyidentified [Pan et al., FEBS Letters, 431:351-356 (1998)]. Aside fromcontaining four putative extracellular domains and a cytoplasmic deathdomain, DR6 is believed to contain a putative leucine-zipper sequencethat overlaps with a proline-rich motif in the cytoplasmic region. Theproline-rich motif resembles sequences that bind to src-homology-3domains, which are found in many intracellular signal-transducingmolecules.

A further group of recently identified receptors are referred to as“decoy receptors,” which are believed to function as inhibitors, ratherthan transducers of signaling. This group includes DCR1 (also referredto as TRID, LIT or TRAIL-R3) [Pan et al., Science, 276:111-113 (1997);Sheridan et al., Science, 277:818-821 (1997); McFarlane et al., J. Biol.Chem., 272:25417-25420 (1997); Schneider et al., FEBS Letters,416:329-334 (1997); Degli-Esposti et al., J. Exp. Med., 186:1165-1170(1997); and Mongkolsapaya et al., J. Immunol., 160:3-6 (1998)] and DCR2(also called TRUNDD or TRAIL-R4) [Marsters et al., Curr. Biol.,7:1003-1006 (1997); Pan et al., FEBS Letters, 424:41-45 (1998);Degli-Esposti et al., Immunity, 7:813-820 (1997)], both cell surfacemolecules, as well as OPG [Simonet et al., supra] and DCR3 [Pitti etal., Nature, 396:699-703 (1998)], both of which are secreted, solubleproteins.

For a review of the TNF family of cytokines and their receptors, seeAshkenazi et al., Science, 281:1305-1308 (1998); Golstein, Curr. Biol.,7:750-753 (1997); and Gruss and Dower, supra.

As presently understood, the cell death program contains at least threeimportant elements—activators, inhibitors, and effectors; in C. elegans,these elements are encoded respectively by three genes, Ced-4, Ced-9 andCed-3 [Steller, Science, 267:1445 (1995); Chinnaiyan et al., Science,275:1122-1126 (1997); Wang et al., Cell, 90:1-20 (1997)]. Two of theTNFR family members, TNFR1 and Fas/Apo1 (CD95), can activate apoptoticcell death [Chinnaiyan and Dixit, Current Biology, 6:555-562 (1996);Fraser and Evan, Cell; 85:781-784 (1996)]. TNFR1 is also known tomediate activation of the transcription factor, NF-KB [Tartaglia et al.,Cell, 74:845-853 (1993); Hsu et al., Cell, 84:299-308 (1996)]. Inaddition to some ECD homology, these two receptors share homology intheir intracellular domain (ICD) in an oligomerization interface knownas the death domain [Tartaglia et al., supra; Nagata, Cell 88:355(1997)]. Death domains are also found in several metazoan proteins thatregulate apoptosis, namely, the Drosophila protein, Reaper, and themammalian proteins referred to as FADD/MORT1, TRADD, and RIP [Cleavelandand Ihle, Cell, 81:479-482 (1995)].

Upon ligand binding and receptor clustering, TNFR1 and CD95 are believedto recruit FADD into a death-inducing signaling complex. CD95purportedly binds FADD directly, while TNFR1 binds FADD indirectly viaTRADD [Chinnaiyan et al., Cell, 81:505-512 (1995); Boldin et al., J.Biol. Chem., 270:387-391 (1995); Hsu et al., supra; Chinnaiyan et al.,J. Biol. Chem., 271:4961-4965 (1996)]. It has been reported that FADDserves as an adaptor protein which recruits the Ced-3-related protease,MACHα/FLICE (caspase 8), into the death signaling complex [Boldin etal., Cell 85:803-815 (1996); Muzio et al., Cell 85:817-827 (1996)].MACHα/FLICE appears to be the trigger that sets off a cascade ofapoptotic proteases, including the interleukin-1β converting enzyme(ICE) and CPP32/Yama, which may execute some critical aspects of thecell death program [Fraser and Evan, supra].

It was recently disclosed that programmed cell death involves theactivity of members of a family of cysteine proteases related to the C.elegans cell death gene, ced-3, and to the mammalian IL-1-convertingenzyme, ICE. The activity of the ICE and CPP32/Yama proteases can beinhibited by the product of the cowpox virus gene, crmA [Ray et al.,Cell 69:597-604 (1992); Tewari et al., Cell, 81:801-809 (1995)]. Recentstudies show that CrmA can inhibit TNFR1- and CD95-induced cell death[Enari et al., Nature, 375:78-81 (1995); Tewari et al., J. Biol. Chem.,270:3255-3260 (1995)].

As reviewed recently by Tewari et al., TNFR1, TNFR2 and CD40 modulatethe expression of proinflammatory and costimulatory cytokines, cytokinereceptors, and cell adhesion molecules through activation of thetranscription factor, NF-KB [Tewari et al., Curr. Op. Genet. Develop.,6:39-44 (1996)]. NF-κB is the prototype of a family of dimerictranscription factors whose subunits contain conserved Rel regions[Verma et al., Genes Develop., 9:2723-2735 (1996); Baldwin, Ann. Rev.Immunol., 14:649-681 (1996)]. In its latent form, NF-κB is complexedwith members of the IκB inhibitor family; upon inactivation of the IκBin response to certain stimuli, released NF-κB translocates to thenucleus where it binds to specific DNA sequences and activates genetranscription.

For other recent reviews of such signaling pathways see, e.g., Ashkenaziet al., Science, 281:1305-1308 (1998) and Nagata, Cell, 88:355-365(1997).

SUMMARY OF THE INVENTION

Applicants have identified cDNA clones that encode novel polypeptideshaving certain sequence identity to previously-described tumor necrosisfactor receptor protein(s), wherein the polypeptides are designated inthe present application as “DNA98853” polypeptide and “DNA101848”polypeptide.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a DNA98853 polypeptide. In certainaspects, the isolated nucleic acid comprises DNA encoding the DNA98853having amino acid residues 1 to 299 or 1 to 136 of FIG. 2 (SEQ ID NO:3),or is complementary to such encoding nucleic acid sequences, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on Apr. 6, 1999 as ATCC 203906which includes the nucleotide sequence encoding DNA98853 polypeptide.

In another embodiment, the invention provides a vector comprising DNAencoding a DNA98853 polypeptide. A host cell comprising such a vector isalso provided. By way of example, the host cells may be CHO cells, E.coli, or yeast. A process for producing DNA98853 polypeptides is furtherprovided and comprises culturing host cells under conditions suitablefor expression of DNA98853 polypeptide and recovering DNA98853polypeptide from the cell culture.

In another embodiment, the invention provides isolated DNA98853polypeptide. In particular, the invention provides isolated nativesequence DNA98853 polypeptide, which in one embodiment, includes anamino acid sequence comprising residues 1 to 299 of FIG. 2 (SEQ IDNO:3). Additional embodiments of the present invention are directed toisolated extracellular domain sequences of a DNA98853 polypeptidecomprising amino acids 1 to 136 of the amino acid sequence shown in FIG.2 (SEQ ID NO:3), or fragments thereof. Optionally, the DNA98853polypeptide is obtained or is obtainable by expressing the polypeptideencoded by the cDNA insert of the vector deposited on Apr. 6, 1999 asATCC 203906.

In another embodiment, the invention provides chimeric moleculescomprising a DNA98853 polypeptide or extracellular domain sequence orother fragment thereof fused to a heterologous polypeptide or amino acidsequence. An example of such a chimeric molecule comprises a DNA98853polypeptide fused to an epitope tag sequence or a Fc region of animmunoglobulin.

In another embodiment, the invention provides an antibody whichspecifically binds to a DNA98853 polypeptide or extracellular domainthereof. Optionally, the antibody is a monoclonal antibody.

In a still further embodiment, the invention provides diagnostic andtherapeutic methods using the DNA98853 polypeptide or DNA encoding theDNA98853 polypeptide.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a DNA101848 polypeptide. In certainaspects, the isolated nucleic acid comprises DNA encoding the DNA101848polypeptide having amino acid residues 1 to 297 or 1 to 136 of FIG. 4(SEQ ID NO:6), or is complementary to such encoding nucleic acidsequences, and remains stably bound to it under at least moderate, andoptionally, under high stringency conditions. The isolated nucleic acidsequence may comprise the cDNA insert of the vector deposited on Apr. 6,1999 as ATCC 203907 which includes the nucleotide sequence encodingDNA101848 polypeptide.

In another embodiment, the invention provides a vector comprising DNAencoding a DNA101848 polypeptide. A host cell comprising such a vectoris also provided. By way of example, the host cells may be CHO cells, E.coli, or yeast. A process for producing DNA101848 polypeptides isfurther provided and comprises culturing host cells under conditionssuitable for expression of DNA101848 polypeptide and recoveringDNA101848 polypeptide from the cell culture.

In another embodiment, the invention provides isolated DNA101848polypeptide. In particular, the invention provides isolated nativesequence DNA101848 polypeptide, which in one embodiment, includes anamino acid sequence comprising residues 1 to 297 of FIG. 4 (SEQ IDNO:6). Additional embodiments of the present invention are directed toisolated extracellular domain sequences of a DNA101848 polypeptidecomprising amino acids 1 to 136 of the amino acid sequence shown in FIG.4 (SEQ ID NO:6), or fragments thereof. Optionally, the DNA101848polypeptide is obtained or is obtainable by expressing the polypeptideencoded by the cDNA insert of the vector deposited on Apr. 6, 1999 asATCC 203907.

In another embodiment, the invention provides chimeric moleculescomprising a DNA101848 polypeptide or extracellular domain sequence orother fragment thereof fused to a heterologous polypeptide or amino acidsequence. An example of such a chimeric molecule comprises a DNA101848polypeptide fused to an epitope tag sequence or a Fc region of animmunoglobulin.

In another embodiment, the invention provides an antibody whichspecifically binds to a DNA101848 polypeptide or extracellular domainthereof. Optionally, the antibody is a monoclonal antibody.

In a still further embodiment, the invention provides diagnostic andtherapeutic methods using the DNA101848 polypeptide or DNA encoding theDNA101848 polypeptide.

Applicants have surprisingly found that the TNF family ligand referredto as EDA-A2 binds to the DNA101848 receptor. The present invention thusprovides for novel methods of using antagonists or agonists of theseTNF-related ligand and receptors. The antagonists and agonists describedherein find utility for, among other things, in vitro, in situ, or invivo diagnosis or treatment of mammalian cells or pathologicalconditions associated with the presence (or absence) of EDA-A2.

The methods of use include methods to treat pathological conditions ordiseases in mammals associated with or resulting from increased orenhanced EDA-A2 expression and/or activity. In the methods of treatment,EDA-A2 antagonists may be administered to the mammal suffering from suchpathological condition or disease. The EDA-A2 antagonists contemplatedfor use in the invention include DNA101848 or DNA98853 receptorimmunoadhesins, as well as antibodies against the DNA101848 or DNA98853receptor, which preferably block or reduce the respective receptorbinding or activation by EDA-A2. The EDA-A2 antagonists contemplated oruse further include anti-EDA-A2 antibodies which are capable of blockingor reducing binding of the ligand to the DNA101848 or DNA98853receptors. Still further antagonist molecules include covalentlymodified forms, or fusion proteins, comprising DNA101848 or DNA98853. Byway of example, such antagonists may include pegylated DNA101848 orDNA98853 or DNA101848 or DNA98853 fused to heterologous sequences suchas epitope tags or leucine zippers.

In another embodiment of the invention, there are provided methods forthe use of EDA-A2 antagonists to block or neutralize the interactionbetween EDA-A2 and DNA101848 or DNA98853. For example, the inventionprovides a method comprising exposing a mammalian cell to one or moreEDA-A2 antagonists in an amount effective to decrease, neutralize orblock activity of the EDA-A2 ligand. The cell may be in cell culture orin a mammal, e.g. a mammal suffering from, for instance, an immunerelated disease or cancer. Thus, the invention includes a method fortreating a mammal suffering from a pathological condition such as animmune related disease or cancer comprising administering an effectiveamount of one or more EDA-A2 antagonists, as disclosed herein.

The invention also provides compositions which comprise one or moreEDA-A2 antagonists. Optionally, the compositions of the invention willinclude pharmaceutically acceptable carriers or diluents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) (and complementarysequence (SEQ ID NO:2)) of a native sequence DNA98853 polypeptide cDNA(nucleotides 1-903). Also presented is the position of threecysteine-rich repeats encoded by nucleotides 10-126, 133-252 and 259-357as underlined. The putative transmembrane domain of the protein isencoded by nucleotides 409-474 in the figure.

FIG. 2 shows the amino acid sequence (SEQ ID NO:3) derived fromnucleotides 1-900 of the nucleotide sequence shown in FIG. 1. Apotential transmembrane domain exists between and including amino acids137 to 158 in the figure.

FIG. 3 shows a nucleotide sequence (SEQ ID NO:4) (and complementarysequence (SEQ ID NO:5)) of a native sequence DNA101848 polypeptide cDNA(nucleotides 1-897). Also presented is the position of threecysteine-rich repeats encoded by nucleotides 10-126, 133-252 and 259-357as underlined. The putative transmembrane domain of the protein isencoded by nucleotides 409-474 in the figure.

FIG. 4 shows the amino acid sequence (SEQ ID NO:6) derived fromnucleotides 1-894 of the nucleotide sequence shown in FIG. 3. Apotential transmembrane domain exists between and including amino acids137 to 158 in the figure.

FIG. 5 illustrates an alignment of the amino acid sequence of aDNA101848 polypeptide (SEQ ID NO:6) with the amino acid sequence of aDNA98853 polypeptide (SEQ ID NO:3). The alignment shows sequenceidentity except for a two amino acid gap in the DNA101848 polypeptide.

FIG. 6 illustrates a schematic representation of a novel inverse longdistance PCR procedure carried out to isolate the full length codingsequence for DNA98853 and DNA101848 polypeptides.

FIG. 7 illustrates Northern Blots showing expression levels of DNA101848polypeptide in several human cell lines and tissues.

FIG. 8 illustrates the results of assays of DNA101848 polypeptide todetermine NF-KB activation. These assays analyze expression of areporter gene driven by a promoter containing a NF-KB responsive elementfrom the E-selectin gene.

FIG. 9 shows the nucleotide sequence of Incyte clone 509 1511H. (SEQ IDNo:7)

FIGS. 10A-10D show the results of an immunostaining assay of MCF-7(transfected cells with N-terminal or C-terminal DNA101848 Flagconstructs) to determine the transmembrane properties of the DNA101848receptor.

FIG. 11 shows the results of an immunostaining assay of COS7 transfectedcells (with various TNF-related ligands) to determine whether DNA101848is a receptor for EDA-A2.

FIGS. 12A-12D show the results of an in situ assay of COS7 cells(transfected with DNA101848 or empty vector). The results showed thatAP-EDA-A2, but not AP-TNF-alpha or AP-TALL-1, bound to the cellstransfected with DNA101848.

FIG. 13 shows the results of a Western blot assay to determine whetherFlag tagged forms of EDA-A2 specifically bind to DNA101848.

FIGS. 14A-14B illustrate the results of assays of DNA101848 and EDA-A2to determine NF-KB activation.

FIGS. 15A-15B illustrate the results of Western blot assays showing theeffects of DNA101848 and EDA-A2 on NF-KB activation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The term “DNA98853 polypeptide” when used herein encompasses nativesequence DNA98853 polypeptide and DNA98853 polypeptide variants (whichare further defined herein). The DNA98853 polypeptides may be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant or synthetic methods.

A “native sequence DNA98853 polypeptide” comprises a polypeptide havingthe same amino acid sequence as a DNA98853 polypeptide derived fromnature. Such native sequence DNA98853 polypeptide can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence DNA98853 polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms of a DNA98853polypeptide (e.g., soluble forms containing for instance, anextracellular domain sequence), naturally-occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants ofa DNA98853 polypeptide. In one embodiment of the invention, the nativesequence DNA98853 polypeptide is a mature or full-length native sequenceDNA98853 polypeptide comprising amino acids 1 to 299 of FIG. 2 (SEQ IDNO:3). In another embodiment of the invention, the native sequenceDNA98853 polypeptide is an extracellular domain sequence of thefull-length DNA98853 polypeptide protein, wherein the putativetransmembrane domain of the full-length DNA98853 polypeptide proteinincludes amino acids 137-158 of the sequence shown in FIG. 2 (SEQ IDNO:3). Thus, additional embodiments of the present invention aredirected to polypeptides comprising amino acids 1-136 of the amino acidsequence shown in FIG. 2 (SEQ ID NO:3). Optionally, the DNA98853polypeptide is obtained or obtainable by expressing the polypeptideencoded by the cDNA insert of the vector DNA98853 deposited on Apr. 6,1999 as ATCC 203906.

The “DNA98853 polypeptide extra cellular domain” or “DNA98853polypeptide ECD” refers to a form of the DNA98853 which is essentiallyfree of the transmembrane and cytoplasmic domains of the DNA98853polypeptide. Ordinarily, DNA98853 polypeptide ECD will have less than 1%of such transmembrane and/or cytoplasmic domains and preferably, willhave less than 0.5% of such domains. Optionally, DNA98853 polypeptideECD will comprise amino acid residues 1-136 of FIG. 2 (SEQ ID NO:3).Included are deletion variants or fragments of the full length or ECD inwhich one or more amino acids are deleted from the N- or C-terminus.Preferably, such deletion variants or fragments possess a desiredactivity, such as described herein. It will be understood that anytransmembrane domain identified for the DNA98853 polypeptide of thepresent invention is identified pursuant to criteria routinely employedin the art for identifying that type of hydrophobic domain. The exactboundaries of a transmembrane domain may vary but most likely by no morethan about 5 amino acids at either end of the domain as initiallyidentified. Accordingly, the DNA98853 polypeptide ECD may optionallycomprise amino acids 1 to X of FIG. 2 (SEQ ID NO:3), wherein X is anyone of amino acid residues 131 to 141 of FIG. 2 (SEQ ID NO:3).

“DNA98853 polypeptide variant” means a DNA98853 polypeptide as definedbelow having at least about 80% amino acid sequence identity with theDNA98853 polypeptide having the deduced amino acid sequence shown inFIG. 2 (SEQ ID NO:3) for a full-length native sequence DNA98853polypeptide or a DNA98853 polypeptide ECD sequence. Such DNA98853polypeptide variants include, for instance, DNA98853 polypeptide whereinone or more amino acid residues are added, or deleted, at the N- orC-terminus of the sequence of FIG. 2 (SEQ ID NO:3). Ordinarily, aDNA98853 polypeptide variant will have at least about 80% amino acidsequence identity, preferably at least about 85% amino acid sequenceidentity, more preferably at least about 90% amino acid sequenceidentity, even more preferably at least about 95% amino acid sequenceidentity and yet more preferably 98% amino acid sequence identity withthe amino acid sequence of FIG. 2 (SEQ ID NO:3).

The term “DNA101848 polypeptide” when used herein encompasses nativesequence DNA101848 polypeptide and DNA101848 polypeptide variants (whichare further defined herein). The DNA101848 polypeptides may be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant or synthetic methods.

A “native sequence DNA101848 polypeptide” comprises a polypeptide havingthe same amino acid sequence as a DNA101848 polypeptide derived fromnature. Such native sequence DNA101848 polypeptide can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence DNA101848 polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms of a DNA101848polypeptide (e.g., soluble forms containing for instance, anextracellular domain sequence), naturally-occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants ofa DNA101848 polypeptide. In one embodiment of the invention, the nativesequence DNA101848 polypeptide is a mature or full-length nativesequence DNA101848 polypeptide comprising amino acids 1 to 297 of FIG. 4(SEQ ID NO:6). In yet another embodiment of the invention, the nativesequence DNA101848 polypeptide is an extracellular domain sequence ofthe full-length DNA101848 polypeptide protein, wherein the putativetransmembrane domain of the full-length DNA101848 polypeptide proteinincludes amino acids 137-158 of the sequence shown in FIG. 4 (SEQ IDNO:6). Thus, additional embodiments of the present invention aredirected to polypeptides comprising amino acids 1-136 of the amino acidsequence shown in FIG. 4 (SEQ ID NO:6). Optionally, the DNA101848polypeptide is obtained or obtainable by expressing the polypeptideencoded by the cDNA insert of the vector DNA101848 deposited on Apr. 6,1999 as ATCC 203907.

The “DNA101848 polypeptide extracellular domain” or “DNA101848polypeptide ECD” refers to a form of the DNA 101848 polypeptide which isessentially free of the transmembrane and cytoplasmic domains of theDNA101848 polypeptide. Ordinarily, DNA101848 polypeptide ECD will haveless than 1% of such transmembrane and/or cytoplasmic domains andpreferably, will have less than 0.5% of such domains. Optionally,DNA101848 polypeptide ECD will comprise amino acid residues 1-136 ofFIG. 4 (SEQ ID NO:6). Included are deletion variants or fragments of thefull length or ECD in which one or more amino acids are deleted from theN- or C-terminus. Preferably, such deletion variants or fragmentspossess a desired activity, such as described herein. It will beunderstood that any transmembrane domain identified for the DNA101848polypeptide of the present invention is identified pursuant to criteriaroutinely employed in the art for identifying that type of hydrophobicdomain. The exact boundaries of a transmembrane domain may vary but mostlikely by no more than about 5 amino acids at either end of the domainas initially identified. Accordingly, the DNA101848 polypeptide ECD mayoptionally comprise amino acids 1 to X of FIG. 4 (SEQ ID NO:6), whereinX is any one of amino acid residues 131 to 141 of FIG. 4 (SEQ ID NO:6).

“DNA101848 polypeptide variant” means a DNA101848 polypeptide as definedbelow having at least about 80% amino acid sequence identity with theDNA101848 polypeptide having the deduced amino acid sequence shown inFIG. 4 (SEQ ID NO:6) for a full-length native sequence DNA101848polypeptide or a DNA101848 polypeptide ECD sequence. Such DNA101848polypeptide variants include, for instance, DNA101848 polypeptideswherein one or more amino acid residues are added, or deleted, at the N-or C-terminus of the sequence of FIG. 4 (SEQ ID NO:6). Ordinarily, aDNA101848 polypeptide variant will have at least about 80% amino acidsequence identity, preferably at least about 85% amino acid sequenceidentity, more preferably at least about 90% amino acid sequenceidentity, even more preferably at least about 95% amino acid sequenceidentity and yet more preferably 98% amino acid sequence identity withthe amino acid sequence of FIG. 4 (SEQ ID NO:6).

“Percent (%) amino acid sequence identity” with respect to thepolypeptide amino acid sequences identified herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in, e.g., a DNA98853 polypeptideor DNA101848 polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Methods for performing sequence alignment anddetermining sequence identity are known to the skilled artisan, may beperformed without undue experimentation, and calculations of identityvalues may be obtained with definiteness. Alignment for purposes ofdetermining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingavailable computer software such as ALIGN or Megalign (DNASTAR)software, WU-BLAST-2 [Altschul et al., Meth. Enzym., 266:460-480(1996)], and ALIGN-2 [authored by Genentech, Inc., and filed with theU.S. Copyright Office on Dec. 10, 1991]. Those skilled in the art candetermine appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximal alignment over the full length ofthe sequences being compared. One may optionally perform the alignmentusing set default parameters in the computer software program.

The term “epitope tagged” where used herein refers to a chimericpolypeptide comprising a DNA98853 polypeptide, or a DNA101848polypeptide, or a domain sequence thereof, fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody may be made, or which can be identified by some otheragent, yet is short enough such that it does not interfere with theactivity of the DNA98853 polypeptide or DNA101848 polypeptide. The tagpolypeptide preferably is also fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 to about 50 amino acid residues (preferably, betweenabout 10 to about 20 residues).

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the DNA98853polypeptide or DNA101848 polypeptide natural environment will not bepresent. Ordinarily, however, isolated polypeptide will be prepared byat least one purification step.

An “isolated” DNA98853 polypeptide-encoding nucleic acid molecule is anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the DNA98853 polypeptide polypeptide-encodingnucleic acid. An isolated DNA98853 polypeptide-encoding nucleic acidmolecule is other than in the form or setting in which it is found innature. Isolated DNA98853 polypeptide-encoding nucleic acid moleculestherefore are distinguished from the DNA98853 polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated DNA98853 polypeptide-encoding nucleic acid molecule includesDNA98853 polypeptide-encoding nucleic acid molecules contained in cellsthat ordinarily express DNA98853 polypeptide where, for example, thenucleic acid molecule is in a chromosomal location different from thatof natural cells.

An “isolated” DNA101848 polypeptide-encoding nucleic acid molecule is anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the DNA101848 polypeptide-encoding nucleicacid. An isolated DNA101848 polypeptide-encoding nucleic acid moleculeis other than in the form or setting in which it is found in nature.Isolated DNA101848 polypeptide-encoding nucleic acid molecules thereforeare distinguished from the DNA101848 polypeptide-encoding nucleic acidmolecule as it exists in natural cells. However, an isolated DNA101848polypeptide-encoding nucleic acid molecule includes DNA101848polypeptide-encoding nucleic acid molecules contained in cells thatordinarily express DNA101848 polypeptide where, for example, the nucleicacid molecule is in a chromosomal location different from that ofnatural cells.

“EDA-A2” or “EDA-A2 ligand” refer to the TNF-related molecule described,e.g, by Bayes et al., Human Molecular Genetics, 7:1661-1669 (1998). Theterms “EDA-A2” or “EDA-A2 polypeptide” when used herein encompass“native sequence EDA-A2 polypeptides” and “EDA-A2 variants”. “EDA-A2” isa designation given to those polypeptides which are encoded by thenucleic acid molecules comprising the polynucleotide sequence shown inBayes et al., supra and variants thereof, nucleic acid moleculescomprising the sequence, and variants thereof as well as fragments ofthe above which have the biological activity (preferably, the ability tobind DNA101848 or DNA98835 receptors) of the native sequence EDA-A2disclosed in Bayes et al., supra. Biologically active variants of EDA-A2will preferably have at least 80%, more preferably, at least 90%, andeven more preferably, at least 95% amino acid sequence identity with thenative sequence EDA-A2 polypeptide described by Bayes et al., supra. A“native sequence” EDA-A2 polypeptide comprises a polypeptide having thesame amino acid sequence as the corresponding EDA-A2 polypeptide derivedfrom nature. Such native sequence EDA-A2 polypeptides can be isolatedfrom nature or can be produced by recombinant and/or synthetic means.The term “native sequence EDA-A2 polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms (e.g., an extracellulardomain sequence), naturally-occurring variant forms (e.g., alternativelyspliced forms) and naturally-occurring allelic variants of thepolypeptide. Applicants did find that the EDA-A1 form of the liganddisclosed in Bayes et al., supra, did not bind Applicants' DNA101848-hFcconstruct (the construct is described in the Examples below), andtherefore, it is believed that that particular EDA-A1 form of the ligandmay not be a biologically active variant for purposes of thisdefinition.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers single anti-DNA98853 polypeptide monoclonal antibodies (includingagonist, antagonist, and neutralizing antibodies), single anti-DNA101848polypeptide monoclonal antibodies (including agonist, antagonist, andneutralizing antibodies), anti-DNA98853 polypeptide antibodycompositions with polyepitopic specificity, and anti-DNA101848polypeptide antibody compositions with polyepitopic specificity. Theterm “monoclonal antibody” as used herein refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts.

“Active” or “activity” for the purposes herein refers to form(s) ofDNA98853 polypeptide which retain the biologic and/or immunologicactivities of native or naturally-occurring DNA98853 polypeptide and toform(s) of DNA101848 polypeptide which retain the biologic and/orimmunologic activities of native or naturally-occurring DNA101848polypeptide. Such activities include, for instance, the ability tomodulate (either in an agonistic or antagonistic manner) apoptosis,proinflammatory or autoimmune responses in mammalian cells, as well asthe ability to bind EDA-A2 ligand. Agonistic activity will include theability to stimulate or enhance an activity, while antagonistic activitywill include the ability to block, suppress or neutralize an activity.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes one ormore biological activities of EDA-A2 polypeptide, in vitro, in situ, orin vivo. Examples of such biological activities of EDA-A2 includebinding of DNA101848 or DNA98853, and activation of NF-KB, as well asthose further reported in the literature.

The term “EDA-A2 antagonist” refers to any molecule that partially orfully blocks, inhibits, or neutralizes a biological activity of EDA-A2,and include, but are not limited to, soluble forms of DNA101848 orDNA98853 receptor such as an extracellular domain sequence of DNA101848or DNA98853, DNA101848 or DNA98853 receptor immunoadhesins, DNA101848 orDNA98853 receptor fusion proteins, covalently modified forms ofDNA101848 or DNA98853 receptor, DNA101848 or DNA98853 variants, andDNA101848 or DNA98853 receptor antibodies. To determine whether anEDA-A2 antagonist molecule partially or fully blocks, inhibits orneutralizes a biological activity of EDA-A2, assays may be conducted toassess the effect(s) of the antagonist molecule on, for example, bindingof EDA-A2 to DNA101848 or DNA98853, or NF-KB activation by therespective ligand. Such assays may be conducted in known in vitro or invivo assay formats, for instance, in transfected cells expressingDNA101848. Preferably, the EDA-A2 antagonist employed in the methodsdescribed herein will be capable of blocking or neutralizing at leastone type of EDA-A2 activity, which may optionally be determined inassays such as described herein. Optionally, an EDA-A2 antagonist willbe capable of reducing or inhibiting binding of EDA-A2 to DNA101848 orDNA98853 by at least 50%, preferably, by at least 90%, more preferablyby at least 99%, and most preferably, by 100%, as compared to a negativecontrol molecule, in a binding assay, such as described in the Examples.

The terms “treating”, “treatment” and “therapy” as used herein refer tocurative therapy, prophylactic therapy, and preventative therapy.

The terms “apoptosis” and “apoptotic activity” are used in a broad senseand refer to the orderly or controlled form of cell death in mammalsthat is typically accompanied by one or more characteristic cellchanges, including condensation of cytoplasm, loss of plasma membranemicrovilli, segmentation of the nucleus, degradation of chromosomal DNAor loss of mitochondrial function. This activity can be determined andmeasured, for instance, by cell viability assays, FACS analysis, or DNAelectrophoresis, all which are known in the art.

The terms “cancer”, “cancerous”, and “malignant” refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth. Examples of cancer include but are notlimited to, carcinoma, including adenocarcinoma, lymphoma, blastoma,melanoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, small-cell lung cancer, non-smallcell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin'slymphoma, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer such as hepatic carcinoma and hepatoma, bladdercancer, breast cancer, colon cancer, colorectal cancer, endometrialcarcinoma, salivary gland carcinoma, kidney cancer such as renal cellcarcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostatecancer, vulval cancer, thyroid cancer, testicular cancer, esophagealcancer, and various types of head and neck cancer.

The term “immune related disease” means a disease in which a componentof the immune system of a mammal causes, mediates or otherwisecontributes to a morbidity in the mammal. Also included are diseases inwhich stimulation or intervention of the immune response has anameliorative effect on progression of the disease. Included within thisterm are autoimmune diseases, immune-mediated inflammatory diseases,non-immune-mediated inflammatory diseases, infectious diseases, andimmunodeficiency diseases. Examples of immune-related and inflammatorydiseases, some of which are immune or T cell mediated, which can betreated according to the invention include systemic lupus erythematosis,rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies,systemic sclerosis (scleroderma), idiopathic inflammatory myopathies(dermatomyositis, polymyositis), Sjogren's syndrome, systemicvasculitis, sarcoidosis, autoimmune hemolytic anemia (immunepancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia), thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),diabetes mellitus, immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis), demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory and fibrotic lung diseases such as inflammatory boweldisease (ulcerative colitis, Crohn's disease), gluten-sensitiveenteropathy, and Whipple's disease, autoimmune or immune-mediated skindiseases including bullous skin diseases, erythema multiforme andcontact dermatitis, psoriasis, allergic diseases such as asthma,allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonias, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, transplantation associated diseases including graftrejection and graft-versus-host-disease. Infectious diseases includeAIDS (HIV infection), hepatitis A, B, C, D, and E, bacterial infections,fungal infections, protozoal infections and parasitic infections.

“Autoimmune disease” is used herein in a broad, general sense to referto disorders or conditions in mammals in which destruction of normal orhealthy tissue arises from humoral or cellular immune responses of theindividual mammal to his or her own tissue constituents. Examplesinclude, but are not limited to, lupus erythematous, thyroiditis,rheumatoid arthritis, psoriasis, multiple sclerosis, autoimmunediabetes, and inflammatory bowel disease (IBD).

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. I¹³¹,I¹²⁵, Y⁹⁰ and R¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of disease. Examples of chemotherapeutic agents includeadriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosinearabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin,taxoids, e.g. paclitaxel (Taxol, Bristol-Myers Squibb Oncology,Princeton, N.J.), and doxetaxel (Taxotere, Rhone-Poulenc Rorer, Antony,Rnace), toxotere, methotrexate, cisplatin, melphalan, CPT-11,vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C,mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide,daunomycin, caminomycin, aminopterin, dactinomycin, mitomycins,esperamicins (see U.S. Pat. No. 4,675,187), melphalan and other relatednitrogen mustards. Also included in this definition are hormonal agentsthat act to regulate or inhibit hormone action such as tamoxifen andonapristone.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, either in vitro or in vivo.Thus, the growth inhibitory agent is one which significantly reduces thepercentage of cells overexpressing such genes in S phase. Examples ofgrowth inhibitory agents include agents that block cell cycleprogression (at a place other than S phase), such as agents that induceG1 arrest and M-phase arrest. Classical M-phase blockers include thevincas (vincristine and vinblastine), taxol, and topo II inhibitors suchas doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest G1 also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogens, and antineoplastic drugs” by Murakami et al (W BSaunders: Philadelphia, 1995), especially p. 13.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; mullerian-inhibiting substance; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors; platelet-growth factor;transforming growth factors (TGFs) such as TGF-alpha and TGF-beta;insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-alpha, -beta, and-gamma; colony stimulating factors (CSFs) such as macrophage-CSF(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF(G-CSF); interleukins (ILs) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; and other polypeptide factors includingLIF and kit ligand (KL). As used herein, the term cytokine includesproteins from natural sources or from recombinant cell culture andbiologically active equivalents of the native sequence cytokines.

The term “mammal” as used herein refers to any mammal classified as amammal, including humans, cows, horses, dogs and cats. In a preferredembodiment of the invention, the mammal is a human.

II. Compositions and Methods of the Invention

A. Full-Length DNA98853 Polypeptide

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas a DNA98853 polypeptide. In particular, Applicants have identified andisolated cDNA encoding a DNA98853 polypeptide, as disclosed in furtherdetail in the Examples below. Using BLAST and FastA sequence alignmentcomputer programs (with set default parameters), Applicants found thatportions of the DNA98853 polypeptide have certain sequence identity withvarious members of the tumor necrosis factor receptor family.Accordingly, it is presently believed that DNA98853 polypeptidedisclosed in the present application is a newly identified member of thetumor necrosis factor receptor family of polypeptides.

The activation of NF-KB by the DNA98853 polypeptide suggests a role forthis protein in modulating apoptosis, proinflammatory and autoimmuneresponses in mammalian cells. It is contemplated for instance, that aDNA98853 polypeptide immunoadhesin molecule (e.g., a DNA98853polypeptide ECD-Ig construct) could be used in an antagonistic manner toblock NF-KB activation.

B. Full-Length DNA101848 Polypeptide

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas a DNA101848 polypeptide. In particular, Applicants have identifiedand isolated cDNA encoding a DNA101848 polypeptide, as disclosed infurther detail in the Examples below. Using BLAST and FastA sequencealignment computer programs (with set default parameters), Applicantsfound that portions of the DNA101848 polypeptide have certain sequenceidentity with various members of the tumor necrosis factor receptorfamily. Accordingly, it is presently believed that DNA101848 polypeptidedisclosed in the present application is a newly identified member of thetumor necrosis factor receptor family of polypeptides.

DNA101848 polypeptide mRNA expression was observed in several cells andtissues. As shown in FIG. 7, relatively high expression levels of DNA101848 polypeptide mRNA were detected in two tumor cell lines, lungcarcinoma A549 and melanoma G361. Relatively weak expression levels werefound in prostate, testis, ovary, thyroid, spinal cord and adrenal glandtissues. Interestingly, a smaller transcript with relatively highexpression level existed in stomach tissue.

The activation of NF-KB by the DNA101848 polypeptide suggests a role forthis protein in modulating apoptosis, proinflammatory and autoimmuneresponses in mammalian cells. It is contemplated for instance, that aDNA101848 polypeptide immunoadhesin molecule (e.g., a DNA101848polypeptide ECD-Ig construct) could be used in an antagonistic manner toblock NF-KB activation.

As described herein, Applicants have found that EDA-A2 acts as a ligandfor DNA101848 receptor. Accordingly, various methods are described foruse of ADA-A2 antagonists. Given the relatively high percentage ofsequence identity between DNA101848 and DNA98853 receptors (particularlythe complete (100%) sequence identity in their respective ECD regions),it is believed that various constructs of DNA98853 may be employed asEDA-A2 antagonists similarly to the antagonistic DNA101848 constructsdescribed herein.

C. Variants of the DNA98853 and DNA101848 Polypeptides

In addition to the full-length native sequence DNA98853 polypeptidedescribed herein, it is contemplated that DNA98853 polypeptide variantscan be prepared. DNA98853 polypeptide variants can be prepared byintroducing appropriate nucleotide changes into the DNA98853polypeptide-encoding DNA, or by synthesis of the desired DNA98853polypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the DNA98853polypeptide, such as changing the number or position of glycosylationsites or altering the membrane anchoring characteristics.

Variations in the native full-length sequence DNA98853 polypeptide or invarious domains of the DNA98853 polypeptide described herein, can bemade, for example, using any of the techniques and guidelines forconservative and non-conservative mutations set forth, for instance, inU.S. Pat. No. 5,364,934. Variations may be a substitution, deletion orinsertion of one or more codons encoding the DNA98853 polypeptide thatresults in a change in the amino acid sequence of the DNA98853polypeptide as compared with the native sequence DNA98853 polypeptide.Optionally, the variation is by substitution of at least one amino acidwith any other amino acid in one or more of the domains of the DNA98853polypeptide.

Similarly, DNA101848 polypeptide variants can be prepared. DNA101848polypeptide variants can be prepared by introducing appropriatenucleotide changes into the DNA101848 polypeptide-encoding DNA, or bysynthesis of the desired DNA101848 polypeptide. Those skilled in the artwill appreciate that amino acid changes may alter post-translationalprocesses of the DNA101848 polypeptide, such as changing the number orposition of glycosylation sites or altering the membrane anchoringcharacteristics.

Variations in the native full-length sequence DNA101848 polypeptide orin various domains of the DNA101848 polypeptide described herein, can bemade, for example, using any of the techniques and guidelines forconservative and non-conservative mutations set forth, for instance, inU.S. Pat. No. 5,364,934. Variations may be a substitution, deletion orinsertion of one or more codons encoding the DNA101848 polypeptide thatresults in a change in the amino acid sequence of the DNA101848polypeptide as compared with the native sequence DNA101848 polypeptide.Optionally, the variation is by substitution of at least one amino acidwith any other amino acid in one or more of the domains of the DNA101848polypeptide.

Guidance in determining which amino acid residue may be inserted,substituted or deleted without adversely affecting the desired activitymay be found by comparing the sequence of the polypeptide with that ofhomologous known protein molecules and minimizing the number of aminoacid sequence changes made in regions of high homology. Amino acidsubstitutions can be the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, such asthe replacement of a leucine with a serine, i.e., conservative aminoacid replacements. Insertions or deletions may optionally be in therange of 1 to 5 amino acids. The variation allowed may be determined bysystematically making insertions, deletions or substitutions of aminoacids in the sequence and testing the resulting variants for activity inany of the in vitro assays described in the Examples below.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the DNA98853 polypeptide or DNA101848polypeptide-encoding variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant. Alanine is alsotypically preferred because it is the most common amino acid. Further,it is frequently found in both buried and exposed positions [Creighton,The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1(1976)]. If alanine substitution does not yield adequate amounts ofvariant, an isosteric amino acid can be used.

D. Modifications of the DNA98853 or DNA101848 Polypeptides

Covalent modifications of DNA98853 polypeptides or of DNA101848polypeptides are included within the scope of this invention. One typeof covalent modification includes reacting targeted amino acid residuesof a DNA98853 polypeptide with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues of a DNA98853 polypeptide. A DNA101848 polypeptide can besimilarly modified at targeted amino acid residues having selected sidechains or at its N- or C-terminal residues.

Derivatization with bifunctional agents is useful, for instance, forcrosslinking DNA98853 polypeptide to a water-insoluble support matrix orsurface for use in the method for purifying anti-DNA98853 polypeptideantibodies, and vice-versa. Such bifunctional agents are also useful forcrosslinking DNA101848 polypeptide to a water-insoluble support matrixor surface for use in the method for purifying anti-DNA101848polypeptide antibodies, and vice-versa. Commonly used crosslinkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the DNA98853 polypeptide orDNA101848 polypeptide included within the scope of this inventioncomprises altering the native glycosylation pattern of eitherpolypeptide. “Altering the native glycosylation pattern” is intended forpurposes herein to mean deleting one or more carbohydrate moieties foundin native sequence DNA98853 polypeptide, deleting one or morecarbohydrate moieties found in native sequence DNA101848 polypeptide,adding one or more glycosylation sites that are not present in thenative sequence DNA98853 polypeptide, and/or adding one or moreglycosylation sites that are not present in the native sequenceDNA101848 polypeptide.

Addition of glycosylation sites to DNA98853 polypeptides or DNA101848polypeptides may be accomplished by altering the amino acid sequencethereof. The alteration may be made, for example, by the addition of, orsubstitution by, one or more serine or threonine residues to the nativesequence DNA98853 polypeptide, or one or more serine or threonineresidues to the native sequence DNA101848 polypeptide (for O-linkedglycosylation sites). The DNA98853 polypeptide amino acid sequence mayoptionally be altered through changes at the DNA level, particularly bymutating the DNA encoding the DNA98853 polypeptide at preselected basessuch that codons are generated that will translate into the desiredamino acids. Similarly, the DNA101848 polypeptide amino acid sequencemay optionally be altered through changes at the DNA level, particularlyby mutating the DNA encoding the DNA101848 polypeptide at preselectedbases such that codons are generated that will translate into thedesired amino acids.

Another means of increasing the number of carbohydrate moieties on theDNA98853 polypeptide or DNA101848 polypeptide is by chemical orenzymatic coupling of glycosides to the polypeptide. Such methods aredescribed in the art, e.g., in WO 87/05330 published 11 Sep. 1987, andin Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the DNA98853 polypeptide orDNA101848 polypeptide may be accomplished chemically or enzymatically orby mutational substitution of codons encoding for amino acid residuesthat serve as targets for glycosylation. Chemical deglycosylationtechniques are known in the art and described, for instance, byHakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edgeet al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage ofcarbohydrate moieties on polypeptides can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura et al.,Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of DNA98853 polypeptide orDNA101848 polypeptide comprises linking the polypeptide to one of avariety of nonproteinaceous polymers, e.g., polyethylene glycol,polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

DNA98853 polypeptides of the present invention may also be modified in away to form chimeric molecules comprising a DNA98853 polypeptide fusedto another, heterologous polypeptide or amino acid sequence. In oneembodiment, such a chimeric molecule comprises a fusion of a DNA98853polypeptide with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the DNA98853 polypeptide.The presence of such epitope-tagged forms of a DNA98853 polypeptide canbe detected using an antibody against the tag polypeptide. Also,provision of the epitope tag enables the DNA98853 polypeptide to bereadily purified by affinity purification using an anti-tag antibody oranother type of affinity matrix that binds to the epitope tag. In analternative embodiment, the chimeric molecule may comprise a fusion of aDNA98853 polypeptide with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule, such afusion could be to the Fc region of an IgG molecule. Optionally, thechimeric molecule will comprise a DNA98853 polypeptide ECD sequencefused to an Fc region of an IgG molecule.

Immunoadhesin molecules are further contemplated for use in the methodsherein. The receptor immunoadhesins may comprise various forms ofDNA101848 or DNA98853, such as the full length polypeptide as well assoluble forms of the receptor which comprise an extracellular domain(ECD) sequence or a fragment of the ECD sequence. In one embodiment, themolecule may comprise a fusion of the DNA101848 or DNA98853 receptorwith an immunoglobulin or a particular region of an immunoglobulin. Fora bivalent form of the immunoadhesin, such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of the receptor polypeptide in place of at least one variableregion within an Ig molecule. In a particularly preferred embodiment,the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions, see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995 and Chamow et al., TIBTECH, 14:52-60 (1996).

The simplest and most straightforward immunoadhesin design combines thebinding domain(s) of the adhesin (e.g. the extracellular domain (ECD) ofa receptor) with the Fc region of an immunoglobulin heavy chain.Ordinarily, when preparing the immunoadhesins of the present invention,nucleic acid encoding the binding domain of the adhesin will be fusedC-terminally to nucleic acid encoding the N-terminus of animmunoglobulin constant domain sequence, however N-terminal fusions arealso possible.

Typically, in such fusions the encoded chimeric polypeptide will retainat least functionally active hinge, C_(H)2 and C_(H)3 domains of theconstant region of an immunoglobulin heavy chain. Fusions are also madeto the C-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the C_(H)1 of the heavy chain or the corresponding regionof the light chain. The precise site at which the fusion is made is notcritical; particular sites are well known and may be selected in orderto optimize the biological activity, secretion, or bindingcharacteristics of the immunoadhesin.

In a preferred embodiment, the adhesin sequence is fused to theN-terminus of the Fc region of immunoglobulin G₁ (IgG₁). It is possibleto fuse the entire heavy chain constant region to the adhesin sequence.However, more preferably, a sequence beginning in the hinge region justupstream of the papain cleavage site which defines IgG Fc chemically(i.e. residue 216, taking the first residue of heavy chain constantregion to be 114), or analogous sites of other immunoglobulins is usedin the fusion. In a particularly preferred embodiment, the adhesin aminoacid sequence is fused to (a) the hinge region and C_(H)2 and C_(H)3 or(b) the C_(H)1, hinge, C_(H)2 and C_(H)3 domains, of an IgG heavy chain.

For bispecific immunoadhesins, the immunoadhesins are assembled asmultimers, and particularly as heterodimers or heterotetramers.Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four chain unit is repeated in the highermolecular weight immunoglobulins; IgM generally exists as a pentamer offour basic units held together by disulfide bonds. IgA globulin, andoccasionally IgG globulin, may also exist in multimeric form in serum.In the case of multimer, each of the four units may be the same ordifferent.

Various exemplary assembled immunoadhesins within the scope herein areschematically diagrammed below:

(a) AC_(L)-AC_(L);

(b) AC_(H)-(AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), orV_(L)C_(L)-AC_(H));

(c) AC_(L)-AC_(H)-(AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), V_(L)C_(L)-AC_(H),or V_(L)C_(L)-V_(H)C_(H))

(d) AC_(L)-V_(H)C_(H)-(AC_(H), or AC_(L)-V_(H)C_(H), orV_(L)C_(L)-AC_(H));

(e) V_(L)C_(L)-AC_(H)-(AC_(L)-V_(H)C_(H), or V_(L)C_(L)-AC_(H)); and

(f) (A-Y)_(n)-(V_(L)C_(L)-V_(H)C_(H))₂,

wherein each A represents identical or different adhesin amino acidsequences;

V_(L) is an immunoglobulin light chain variable domain;

V_(H) is an immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H) is an immunoglobulin heavy chain constant domain;

n is an integer greater than 1;

Y designates the residue of a covalent cross-linking agent.

In the interests of brevity, the foregoing structures only show keyfeatures; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown. However, where suchdomains are required for binding activity, they shall be constructed tobe present in the ordinary locations which they occupy in theimmunoglobulin molecules.

Alternatively, the adhesin sequences can be inserted betweenimmunoglobulin heavy chain and light chain sequences, such that animmunoglobulin comprising a chimeric heavy chain is obtained. In thisembodiment, the adhesin sequences are fused to the 3′ end of animmunoglobulin heavy chain in each arm of an immunoglobulin, eitherbetween the hinge and the C_(H)2 domain, or between the C_(H)2 andC_(H)3 domains. Similar constructs have been reported by Hoogenboom etal., Mol. Immunol., 28:1027-1037 (1991).

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to anadhesin-immunoglobulin heavy chain fusion polypeptide, or directly fusedto the adhesin. In the former case, DNA encoding an immunoglobulin lightchain is typically coexpressed with the DNA encoding theadhesin-immunoglobulin heavy chain fusion protein. Upon secretion, thehybrid heavy chain and the light chain will be covalently associated toprovide an immunoglobulin-like structure comprising two disulfide-linkedimmunoglobulin heavy chain-light chain pairs. Methods suitable for thepreparation of such structures are, for example, disclosed in U.S. Pat.No. 4,816,567, issued 28 Mar. 1989.

Immunoadhesins are most conveniently constructed by fusing the cDNAsequence encoding the adhesin portion in-frame to an immunoglobulin cDNAsequence. However, fusion to genomic immunoglobulin fragments can alsobe used (see, e.g. Aruffo et al., Cell, 61: 1303-1313 (1990); andStamenkovic et al., Cell, 66:1133-1144 (1991)). The latter type offusion requires the presence of Ig regulatory sequences for expression.cDNAs encoding IgG heavy-chain constant regions can be isolated based onpublished sequences from cDNA libraries derived from spleen orperipheral blood lymphocytes, by hybridization or by polymerase chainreaction (PCR) techniques. The cDNAs encoding the “adhesin” and theimmunoglobulin parts of the immunoadhesin are inserted in tandem into aplasmid vector that directs efficient expression in the chosen hostcells.

DNA101848 polypeptides of the present invention may also be modified ina way to form chimeric molecules comprising a DNA101848 polypeptidefused to another, heterologous polypeptide or amino acid sequence. Inone embodiment, such a chimeric molecule comprises a fusion of aDNA101848 polypeptide with a tag polypeptide which provides an epitopeto which an anti-tag antibody can selectively bind. The epitope tag isgenerally placed at the amino- or carboxyl-terminus of the DNA101848polypeptide. The presence of such epitope-tagged forms of a DNA101848polypeptide can be detected using an antibody against the tagpolypeptide. Also, provision of the epitope tag enables the DNA101848polypeptide to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. In an alternative embodiment, the chimeric molecule maycomprise a fusion of a DNA101848 polypeptide with an immunoglobulin or aparticular region of an immunoglobulin. For a bivalent form of thechimeric molecule, such a fusion could be to the Fc region of an IgGmolecule. Optionally, the chimeric molecule will comprise a DNA101848polypeptide ECD sequence fused to an Fc region of an IgG molecule.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem. 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

The DNA98853 polypeptide of the present invention may also be modifiedin a way to form a chimeric molecule comprising a DNA98853 polypeptidefused to a leucine zipper. Similarly, the DNA101848 polypeptide of thepresent invention may also be modified in a way to form a chimericmolecule comprising a DNA101848 polypeptide fused to a leucine zipper.Various leucine zipper polypeptides have been described in the art. See,e.g., Landschulz et al., Science 240:1759 (1988); WO 94/10308; Hoppe etal., FEBS Letters 344:1991 (1994); Maniatis et al., Nature 341:24(1989). It is believed that use of a leucine zipper fused to a DNA98853polypeptide may be desirable to assist in dimerizing or trimerizingsoluble DNA98853 polypeptide in solution, and that a leucine zipperfused to a DNA101848 polypeptide may be desirable to assist indimerizing or trimerizing soluble DNA101848 polypeptide in solution.Those skilled in the art will appreciate that the leucine zipper may befused at either the N- or C-terminal end of the DNA98853 or DNA101848polypeptide molecule.

D. Preparation of Polypeptides

1. Preparation of DNA98853 Polypeptide

The description below relates primarily to production of a polypeptide,such as DNA98853 polypeptide, by culturing cells transformed ortransfected with a vector containing DNA98853 polypeptide encodingnucleic acid. It is, of course, contemplated that alternative methods,which are well known in the art, may be employed to prepare DNA98853polypeptides. For instance, the DNA98853 polypeptide sequence, orportions thereof, may be produced by direct peptide synthesis usingsolid-phase techniques [see, e.g., Stewart et al., Solid-Phase PeptideSynthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield,J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro protein synthesis maybe performed using manual techniques or by automation. Automatedsynthesis may be accomplished, for instance, using an Applied BiosystemsPeptide Synthesizer (Foster City, Calif.) using manufacturer'sinstructions. Various portions of DNA98853 polypeptides may bechemically synthesized separately and combined using chemical orenzymatic methods to produce a full-length DNA98853 polypeptide.

2. Preparation of DNA101848 Polypeptide

The description below also relates to production of DNA101848polypeptide by culturing cells transformed or transfected with a vectorcontaining DNA101848 polypeptide encoding nucleic acid. It is, ofcourse, contemplated that alternative methods, which are well known inthe art, may be employed to prepare DNA101848 polypeptides. Forinstance, the DNA101848 polypeptide sequence, or portions thereof, maybe produced by direct peptide synthesis using solid-phase techniques, asdescribed above. Various portions of DNA101848 polypeptides may bechemically synthesized separately and combined using chemical orenzymatic methods to produce a full-length DNA101848 polypeptide.

3. Isolation of DNA Encoding the DNA98853 or DNA101848 Polypeptides

DNA encoding a DNA98853 polypeptide may be obtained from a cDNA libraryprepared from tissue believed to possess the DNA98853 polypeptide mRNAand to express it at a detectable level. Accordingly, human DNA98853polypeptide-encoding DNA can be conveniently obtained from a cDNAlibrary prepared from human tissue, such as described in the Examples.The DNA98853 polypeptide-encoding gene may also be obtained from agenomic library or by oligonucleotide synthesis.

Similarly, DNA encoding a DNA101848 polypeptide may be obtained from acDNA library prepared from tissue believed to possess the DNA101848polypeptide mRNA and to express it at a detectable level. Accordingly,human DNA101848 polypeptide-encoding DNA can be conveniently obtainedfrom a cDNA library prepared from human tissue, such as described in theExamples. The DNA101848 polypeptide-encoding gene may also be obtainedfrom a genomic library or by oligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies to a DNA98853polypeptide, antibodies to a DNA101848 polypeptide, or oligonucleotidesof at least about 20-80 bases) designed to identify the gene of interestor the protein encoded by it. Screening the cDNA or genomic library withthe selected probe may be conducted using standard procedures, such asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual(New York: Cold Spring Harbor Laboratory Press, 1989). An alternativemeans to isolate the gene encoding DNA98853 polypeptide or the geneencoding DNA101848 polypeptide is to use PCR methodology [Sambrook etal., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (ColdSpring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined through sequence alignment using computer software programssuch as ALIGN, DNAstar, and INHERIT.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

4. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for DNA98853 polypeptide production.Alternatively, host cells are transfected or transformed with expressionor cloning vectors described herein for DNA101848 polypeptideproduction. The host cells are cultured in conventional nutrient mediamodified as appropriate for inducing promoters, selecting transformants,or amplifying the genes encoding the desired sequences. The cultureconditions, such as media, temperature, pH and the like, can be selectedby the skilled artisan without undue experimentation. In general,principles, protocols, and practical techniques for maximizing theproductivity of cell cultures can be found in Mammalian CellBiotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991)and Sambrook et al., supra.

Methods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Depending on the host cell used,transformation is performed using standard techniques appropriate tosuch cells. The calcium treatment employing calcium chloride, asdescribed in Sambrook et al., supra, or electroporation is generallyused for prokaryotes or other cells that contain substantial cell-wallbarriers. Infection with Agrobacterium tumefaciens is used fortransformation of certain plant cells, as described by Shaw et al.,Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact. 130:946 (1977) and Hsiao et al.,Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods forintroducing DNA into cells, such as by nuclear microinjection,electroporation, bacterial protoplast fusion with intact cells, orpolycations, e.g., polybrene, polyornithine, may also be used. Forvarious techniques for transforming mammalian cells, see Keown et al.,Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature,336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coliX1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635).

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for vectorsencoding DNA98853 polypeptide or vectors encoding DNA101848 polypeptide.Saccharomyces cerevisiae is a commonly used lower eukaryotic hostmicroorganism.

Suitable host cells for the expression of glycosylated DNA98853polypeptide or of glycosylated DNA101848 polypeptide are derived frommulticellular organisms. Examples of invertebrate cells include insectcells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells.Examples of useful mammalian host cell lines include Chinese hamsterovary (CHO) and COS cells. More specific examples include monkey kidneyCV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonickidney line (293 or 293 cells subcloned for growth in suspensionculture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamsterovary cells/−DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)); human lung cells (WI 38, ATCC CCL 75); human livercells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCCCCL51). The selection of the appropriate host cell is deemed to bewithin the skill in the art.

5. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding the desiredDNA98853 polypeptide or encoding the desired DNA101848 polypeptide maybe inserted into a replicable vector for cloning (amplification of theDNA) or for expression. Various vectors are publicly available. Thevector may, for example, be in the form of a plasmid, cosmid, viralparticle, or phage. The appropriate nucleic acid sequence may beinserted into the vector by a variety of procedures. In general, DNA isinserted into an appropriate restriction endonuclease site(s) usingtechniques known in the art. Vector components generally include, butare not limited to, one or more of a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence. Construction of suitablevectors containing one or more of these components employs standardligation techniques which are known to the skilled artisan.

The desired DNA98853 polypeptide or the desired DNA101848 polypeptidemay be produced recombinantly not only directly, but also as a fusionpolypeptide with a heterologous polypeptide, which may be a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide. In general, the signalsequence may be a component of the vector, it may be a part of theDNA98853 polypeptide-encoding DNA that is inserted into the vector, orit may be a part of the DNA101848 polypeptide-encoding DNA that isinserted into the vector. The signal sequence may be a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces α-factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression, mammaliansignal sequences may be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up theDNA98853 polypeptide-encoding nucleic acid or the DNA101848polypeptide-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10: 157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the DNA98853 polypeptide-encoding nucleic acid sequence or tothe DNA101848 polypeptide-encoding nucleic acid sequence. The promoterdirects mRNA synthesis. Promoters recognized by a variety of potentialhost cells are well known. Promoters suitable for use with prokaryotichosts include the β-lactamase and lactose promoter systems [Chang etal., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters suchas the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25(1983)]. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theDNA98853 polypeptide or operably linked to the DNA encoding theDNA101848 polypeptide.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem. 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phos-phate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

DNA98853 polypeptide or DNA101848 polypeptide transcription from vectorsin mammalian host cells is controlled, for example, by promotersobtained from the genomes of viruses such as polyoma virus, fowlpoxvirus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, and from heat-shock promoters, providedsuch promoters are compatible with the host cell systems.

Transcription by higher eukaryotes of a DNA encoding a DNA98853polypeptide or of a DNA encoding a DNA101848 polypeptide may beincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, thatact on a promoter to increase its transcription. Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theDNA98853 polypeptide coding sequence, but is preferably located at asite 5′ from the promoter. Similarly, the enhancer may be spliced intothe vector at a position 5′ or 3′ to the DNA101848 polypeptide codingsequence, but is preferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding DNA98853 polypeptide or of the mRNAencoding DNA101848 polypeptide.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of DNA98853 polypeptides and/or DNA101848 polypeptides inrecombinant vertebrate cell culture are described in Gething et al.,Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP117,060; and EP 117,058.

6. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceDNA98853 polypeptide, against a native sequence DNA101848 polypeptide,against a synthetic peptide based on the DNA sequences provided herein,against an exogenous sequence fused to DNA98853 polypeptide-encoding DNAand encoding a specific antibody epitope, or against an exogenoussequence fused to DNA101848 polypeptide-encoding DNA and encoding aspecific antibody epitope.

7. Polypeptide Purification

Forms of DNA98853 polypeptide or DNA101848 polypeptide may be recoveredfrom culture medium or from host cell lysates. If membrane-bound, theycan be released from the membrane using a suitable detergent solution(e.g. Triton-X 100) or by enzymatic cleavage. Cells employed inexpression of DNA98853 polypeptides or DNA101848 polypeptides can bedisrupted by various physical or chemical means, such as freeze-thawcycling, sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify DNA98853 polypeptide or DNA101848polypeptide from recombinant cell proteins or polypeptides. Thefollowing procedures are exemplary of suitable purification procedures:by fractionation on an ion-exchange column; ethanol precipitation;reverse phase HPLC; chromatography on silica or on a cation-exchangeresin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfateprecipitation; gel filtration using, for example, Sephadex G-75; proteinA Sepharose columns to remove contaminants such as IgG; and metalchelating columns to bind epitope-tagged forms of the DNA98853polypeptide or DNA101848 polypeptide. Various methods of proteinpurification may be employed and such methods are known in the art anddescribed for example in Deutscher, Methods in Enzymology, 182 (1990);Scopes, Protein Purification: Principles and Practice, Springer-Verlag,New York (1982). The purification step(s) selected will depend, forexample, on the nature of the production process used and the particularDNA98853 polypeptide or DNA101848 polypeptide produced.

E. Uses for DNA98853 Polypeptide or DNA101848 Polypeptide

Nucleotide sequences (or their complement) encoding DNA98853polypeptides, and nucleotide sequences or their complements encodingDNA101848 polypeptides, have various applications in the art ofmolecular biology, including uses as hybridization probes, in chromosomeand gene mapping and in the generation of anti-sense RNA and DNA.DNA98853 polypeptide-encoding nucleic acid will also be useful for thepreparation of DNA98853 polypeptides by the recombinant techniquesdescribed herein. Similarly, DNA101848 polypeptide-encoding nucleic acidwill also be useful for the preparation of DNA101848 polypeptides by therecombinant techniques described herein.

The full-length DNA98853 nucleotide sequence (SEQ ID NO:1) or thefull-length native sequence DNA98853 polypeptide (SEQ ID NO:3) sequence,or portions thereof, may be used as hybridization probes for a cDNAlibrary to isolate the full-length DNA98853 polypeptide gene or toisolate still other genes (for instance, those encodingnaturally-occurring variants of DNA98853 polypeptide or DNA98853polypeptide from other species) which have a desired sequence identityto the DNA98853 polypeptide nucleotide sequence disclosed in FIG. 1 (SEQID NO:1). Optionally, the length of the probes will be about 20 to about50 bases. The hybridization probes may be derived from the DNA98853nucleotide sequence of SEQ ID NO:1 as shown in FIG. 1 or from genomicsequences including promoters, enhancer elements and introns of nativesequence DNA98853 polypeptide-encoding DNA. By way of example, ascreening method will comprise isolating the coding region of theDNA98853 polypeptide gene using the known DNA sequence to synthesize aselected probe of about 40 bases.

Similarly, the full-length DNA101848 nucleotide sequence (SEQ ID NO:4)or the full-length native sequence DNA101848 polypeptide (SEQ ID NO:6)sequence, or portions thereof, may be used as hybridization probes for acDNA library to isolate the full-length DNA101848 polypeptide gene or toisolate still other genes (for instance, those encodingnaturally-occurring variants of DNA101848 polypeptide or DNA101848polypeptide from other species) which have a desired sequence identityto the DNA101848 polypeptide sequence disclosed in FIG. 4 (SEQ ID NO:6).Optionally, the length of the probes will be about 20 to about 50 bases.The hybridization probes may be derived from the DNA101848 nucleotidesequence of SEQ ID NO:4 as shown in FIG. 3 or from genomic sequencesincluding promoters, enhancer elements and introns of native sequenceDNA101848 polypeptide-encoding DNA. By way of example, a screeningmethod will comprise isolating the coding region of the DNA101848polypeptide gene using the known DNA sequence to synthesize a selectedprobe of about 40 bases.

Hybridization probes may be labeled by a variety of labels, includingradionucleotides such as ³²P or ³⁵S, or enzymatic labels such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems. Labeled probes having a sequence complementary to that of theDNA98853 polypeptide gene of the present invention, or complementary tothat of the DNA101848 polypeptide gene of the present invention, can beused to screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related DNA98853 polypeptidesequences or DNA101848 polypeptide sequences.

Nucleotide sequences encoding a DNA98853 polypeptide or a DNA101848polypeptide can also be used to construct hybridization probes formapping the gene which encodes that polypeptide, and for the geneticanalysis of individuals with genetic disorders. The nucleotide sequencesprovided herein may be mapped to a chromosome and specific regions of achromosome using known techniques, such as in situ hybridization,linkage analysis against known chromosomal markers, and hybridizationscreening with libraries.

When the coding sequences for DNA98853 polypeptide encode a proteinwhich binds to another protein (example, where the DNA98853 polypeptidefunctions as a receptor), the DNA98853 polypeptide can be used in assaysto identify the other proteins or molecules involved in the bindinginteraction. Similarly, when the coding sequences for DNA101848polypeptide encode a protein which binds to another protein (example,where the DNA101848 polypeptide functions as a receptor), the DNA101848polypeptide can be used in assays to identify the other proteins ormolecules involved in the binding interaction.

By such methods, inhibitors of the receptor/ligand binding interactioncan be identified. Proteins involved in such binding interactions canalso be used to screen for peptide or small molecule inhibitors oragonists of the binding interaction. Also, the receptor DNA98853polypeptide or the receptor DNA101848 polypeptide can be used to isolateother correlative ligand(s). Screening assays can be designed to findlead compounds that mimic the biological activity of a native DNA98853polypeptide, a native DNA101848 polypeptide, a receptor for DNA98853polypeptide, or a receptor for DNA101848 polypeptide. Such screeningassays will include assays amenable to high-throughput screening ofchemical libraries, making them particularly suitable for identifyingsmall molecule drug candidates. Small molecules contemplated includesynthetic organic or inorganic compounds. The assays can be performed ina variety of formats, including protein-protein binding assays,biochemical screening assays, immunoassays and cell based assays, whichare well characterized in the art.

Nucleic acids which encode DNA98853 polypeptide, DNA101848 polypeptide,or any of their modified forms can also be used to generate eithertransgenic animals or “knock out” animals which, in turn, are useful inthe development and screening of therapeutically useful reagents. Atransgenic animal (e.g., a mouse or rat) is an animal having cells thatcontain a transgene, which transgene was introduced into the animal oran ancestor of the animal at a prenatal, e.g., an embryonic stage. Atransgene is a DNA which is integrated into the genome of a cell fromwhich a transgenic animal develops. In one embodiment, cDNA encodingDNA98853 polypeptide can be used to clone genomic DNA encoding DNA98853polypeptide in accordance with established techniques and the genomicsequences used to generate transgenic animals that contain cells whichexpress DNA encoding DNA98853 polypeptide. In another embodiment, cDNAencoding DNA101848 polypeptide can be used to clone genomic DNA encodingDNA101848 polypeptide in accordance with established techniques and thegenomic sequences used to generate transgenic animals that contain cellswhich express DNA encoding DNA101848 polypeptide.

Methods for generating transgenic animals, particularly animals such asmice or rats, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically,particular cells would be targeted for DNA98853 polypeptide and/orDNA101848 polypeptide transgene incorporation with tissue-specificenhancers. Transgenic animals that include a copy of a transgeneencoding DNA98853 polypeptide introduced into the germ line of theanimal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding DNA98853 polypeptide.Alternatively, transgenic animals that include a copy of a transgeneencoding DNA101848 polypeptide introduced into the germ line of theanimal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding DNA101848 polypeptide. Such animalscan be used as tester animals for reagents thought to confer protectionfrom, for example, pathological conditions associated with itsoverexpression. In accordance with this facet of the invention, ananimal is treated with the reagent and a reduced incidence of thepathological condition, compared to untreated animals bearing thetransgene, would indicate a potential therapeutic intervention for thepathological condition.

Alternatively, non-human homologues of DNA98853 polypeptide can be usedto construct a DNA98853 polypeptide “knock out” animal which has adefective or altered gene encoding DNA98853 polypeptide as a result ofhomologous recombination between the endogenous gene encoding DNA98853polypeptide and altered genomic DNA encoding DNA98853 polypeptideintroduced into an embryonic cell of the animal. For example, cDNAencoding DNA98853 polypeptide can be used to clone genomic DNA encodingDNA98853 polypeptide in accordance with established techniques. Aportion of the genomic DNA encoding DNA98853 polypeptide can be deletedor replaced with another gene, such as a gene encoding a selectablemarker which can be used to monitor integration.

Similarly, non-human homologues of DNA101848 polypeptide can be used toconstruct a DNA101848 polypeptide “knock out” animal which has adefective or altered gene encoding DNA101848 polypeptide as a result ofhomologous recombination between the endogenous gene encoding DNA101848polypeptide and altered genomic DNA encoding DNA101848 polypeptideintroduced into an embryonic cell of the animal. For example, cDNAencoding DNA101848 polypeptide can be used to clone genomic DNA encodingDNA101848 polypeptide in accordance with established techniques. Aportion of the genomic DNA encoding DNA101848 polypeptide can be deletedor replaced with another gene, such as a gene encoding a selectablemarker which can be used to monitor integration.

Typically, in constructing a “knock out animal”, several kilobases ofunaltered flanking DNA (both at the 5′ and 3′ ends) are included in thevector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for adescription of homologous recombination vectors]. The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced DNA has homologously recombined withthe endogenous DNA are selected [see e.g., Li et al., Cell 69:915(1992)]. The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse or rat) to form aggregation chimeras [see e.g.,Bradley, in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term to create a “knockout” animal. Progeny harboring the homologously recombined DNA in theirgerm cells can be identified by standard techniques and used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA. Knock out animals can be characterized for instance, fortheir ability to defend against certain pathological conditions and fortheir development of pathological conditions due to absence of theDNA98853 polypeptide or the DNA101848 polypeptide.

The DNA98853 polypeptide or the DNA101848 polypeptide herein may beemployed in accordance with the present invention by expression of suchpolypeptides in vivo, which is often referred to as gene therapy.

There are two major approaches to getting the nucleic acid (optionallycontained in a vector) into the patient's cells: in vivo and ex vivo.For in vivo delivery the nucleic acid is injected directly into thepatient, usually at the sites where the polypeptide is required. Forexample, DNA98853 polypeptide-encoding nucleic acid will be injected atthe site of synthesis of the DNA98853 polypeptide, if known, or the sitewhere biological activity of DNA98853 polypeptide is needed. Forexample, DNA101848 polypeptide-encoding nucleic acid will be injected atthe site of synthesis of the DNA101848 polypeptide, if known, or thesite where biological activity of DNA101848 polypeptide is needed. Forex vivo treatment, the patient's cells are removed, the nucleic acid isintroduced into these isolated cells, and the modified cells areadministered to the patient either directly or, for example,encapsulated within porous membranes that are implanted into the patient(see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187).

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or transferredin vivo in the cells of the intended host. Techniques suitable for thetransfer of nucleic acid into mammalian cells in vitro include the useof liposomes, electroporation, microinjection, transduction, cellfusion, DEAE-dextran, the calcium phosphate precipitation method, etc.Transduction involves the association of a replication-defective,recombinant viral (preferably retroviral) particle with a cellularreceptor, followed by introduction of the nucleic acids contained by theparticle into the cell. A commonly used vector for ex vivo delivery ofthe gene is a retrovirus.

The currently preferred in vivo nucleic acid transfer techniques includetransfection with viral or non-viral vectors (such as adenovirus,lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV)) andlipid-based systems (useful lipids for lipid-mediated transfer of thegene are, for example, DOTMA, DOPE, and DC-Chol; see, e.g., Tonkinson etal., Cancer Investigation, 14(1): 54-65 (1996)). The most preferredvectors for use in gene therapy are viruses, most preferablyadenoviruses, AAV, lentiviruses, or retroviruses. A viral vector such asa retroviral vector includes at least one transcriptionalpromoter/enhancer or locus-defining element(s), or other elements thatcontrol gene expression by other means such as alternate splicing,nuclear RNA export, or post-translational modification of messenger. Inaddition, a viral vector such as a retroviral vector includes a nucleicacid molecule that, when transcribed in the presence of a gene encodingDNA98853 polypeptide or of a gene encoding DNA101848 polypeptide, isoperably linked thereto and acts as a translation initiation sequence.Such vector constructs also include a packaging signal, long terminalrepeats (LTRs) or portions thereof, and positive and negative strandprimer binding sites appropriate to the virus used (if these are notalready present in the viral vector). In addition, such vector typicallyincludes a signal sequence for secretion of the DNA98853 polypeptide orDNA101848 polypeptide from a host cell in which it is placed. Preferablythe signal sequence for this purpose is a mammalian signal sequence,most preferably the native signal sequence for DNA98853 polypeptide orfor DNA101848 polypeptide. Optionally, the vector construct may alsoinclude a signal that directs polyadenylation, as well as one or morerestriction sites and a translation termination sequence. By way ofexample, such vectors will typically include a 5′ LTR, a tRNA bindingsite, a packaging signal, an origin of second-strand DNA synthesis, anda 3′ LTR or a portion thereof. Other vectors can be used that arenon-viral, such as cationic lipids, polylysine, and dendrimers.

In some situations, it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell-surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins that bind to a cell-surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins that undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem., 262: 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA, 87: 3410-3414 (1990). For a review of the currentlyknown gene marking and gene therapy protocols, see Anderson et al.,Science, 256: 808-813 (1992). See also WO 93/25673 and the referencescited therein.

Suitable gene therapy and methods for making retroviral particles andstructural proteins can be found in, e.g., U.S. Pat. No. 5,681,746.

DNA98853 polypeptides or DNA101848 polypeptides of the present inventionwhich possess biological activity, for example such as related to thatof the known tumor necrosis factor receptors may be employed both invivo for therapeutic purposes and in vitro.

Therapeutic compositions of the DNA98853 polypeptide or the DNA101848polypeptide can be prepared by mixing the desired molecule having theappropriate degree of purity with optional pharmaceutically acceptablecarriers, excipients, or stabilizers (Remington's PharmaceuticalSciences, 16th edition, Oslo, A. ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are preferably nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

Additional examples of such carriers include ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts, or electrolytes such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, and polyethylene glycol.Carriers for topical or gel-based forms of include polysaccharides suchas sodium carboxymethylcellulose or methylcellulose,polyvinylpyrrolidone, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol,and wood wax alcohols. For all administrations, conventional depot formsare suitably used. Such forms include, for example, microcapsules,nano-capsules, liposomes, plasters, inhalation forms, nose sprays,sublingual tablets, and sustained-release preparations. The DNA98853polypeptides or DNA101848 polypeptides will typically be formulated insuch vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml.

DNA98853 polypeptide or DNA101848 polypeptide to be used for in vivoadministration should be sterile. This is readily accomplished byfiltration through sterile filtration membranes, prior to or followinglyophilization and reconstitution. DNA98853 polypeptide or DNA101848polypeptide ordinarily will be stored in lyophilized form or in solutionif administered systemically. If in lyophilized form, DNA98853polypeptide or DNA101848 polypeptide is typically formulated incombination with other ingredients for reconstitution with anappropriate diluent at the time for use. An example of a liquidformulation of DNA98853 polypeptide or DNA101848 polypeptide is asterile, clear, colorless unpreserved solution filled in a single-dosevial for subcutaneous injection.

Therapeutic DNA98853 polypeptide or DNA101848 polypeptide compositionsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle. The formulations are preferablyadministered as repeated intravenous (i.v.), subcutaneous (s.c.), orintramuscular (i.m.) injections, or as aerosol formulations suitable forintranasal or intrapulmonary delivery (for intrapulmonary delivery see,e.g., EP 257,956).

DNA98853 polypeptide or DNA101848 polypeptide can also be administeredin the form of sustained-released preparations. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the protein, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (e.g.,poly(2-hydroxyethyl-methacrylate) as described by Langer et al., J.Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982) or poly(vinylalcohol)), polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556 (1983)),non-degradable ethylene-vinyl acetate (Langer et al., supra), degradablelactic acid-glycolic acid copolymers such as the Lupron Depot(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid (EP133,988).

The therapeutically effective dose of a DNA98853 polypeptide or aDNA101848 polypeptide (or antibody thereto) will, of course, varydepending on such factors as the intended therapy (e.g., for modulatingapoptosis, autoimmune or proinflammatory responses), the pathologicalcondition to be treated, the method of administration, the type ofcompound being used for treatment, any co-therapy involved, thepatient's age, weight, general medical condition, medical history, etc.,and its determination is well within the skill of a practicingphysician. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe maximal therapeutic effect.

With the above guidelines, the effective dose generally is within therange of from about 0.001 to about 1.0 mg/kg.

The route of administration of DNA98853 polypeptide or DNA101848polypeptide is in accord with known methods, e.g., by injection orinfusion by intravenous, intramuscular, intracerebral, intraperitoneal,intracerobrospinal, subcutaneous, intraocular, intraarticular,intrasynovial, intrathecal, oral, topical, or inhalation routes, or bysustained-release systems. The DNA98853 polypeptide or DNA101848polypeptide also are suitably administered by intratumoral, peritumoral,intralesional, or perilesional routes, to exert local as well assystemic therapeutic effects.

The effectiveness of the DNA98853 polypeptide or DNA101848 polypeptidetreating the disorder may be improved by administering the active agentserially or in combination with another agent that is effective forthose purposes, either in the same composition or as separatecompositions. Examples of such agents include cytotoxic,chemotherapeutic or growth-inhibitory agents, cytokines and radiologicaltreatments (such as involving irradiation or administration ofradiological substances).

The effective amounts of the therapeutic agents administered incombination with DNA98853 polypeptide or DNA101848 polypeptide will beat the physician's discretion. Dosage administration and adjustment isdone to achieve maximal management of the conditions to be treated.

The various therapeutic methods and compositions referred to above maybe similarly employed for use of the EDA-A2 antagonists describedherein.

F. Anti-DNA98853 Polypeptide and/or Anti-DNA101848 PolypeptideAntibodies

The present invention further provides anti-DNA98853 polypeptideantibodies and anti-DNA101848 polypeptide antibodies. Exemplaryantibodies include polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-DNA98853 polypeptide antibodies and anti-DNA101848 polypeptideantibodies of the present invention may comprise polyclonal antibodies.Methods of preparing polyclonal antibodies are known to the skilledartisan. Polyclonal antibodies can be raised in a mammal, for example,by one or more injections of an immunizing agent and, if desired, anadjuvant. Typically, the immunizing agent and/or adjuvant will beinjected in the mammal by multiple subcutaneous or intraperitonealinjections. For anti-DNA98853 polypeptide antibodies, the immunizingagent may include the DNA98853 polypeptide or a fusion protein thereof.For anti-DNA101848 polypeptide antibodies, the immunizing agent mayinclude the DNA101848 polypeptide or a fusion protein thereof. It may beuseful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized. Examples of such immunogenicproteins include but are not limited to keyhole limpet hemocyanin, serumalbumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examplesof adjuvants which may be employed include Freund's complete adjuvantand MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalosedicorynomycolate). The immunization protocol may be selected by oneskilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-DNA98853 polypeptide antibodies or anti-DNA101848 polypeptideantibodies may, alternatively, be monoclonal antibodies. Monoclonalantibodies may be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein, Nature, 256:495 (1975). In a hybridomamethod, a mouse, hamster, or other appropriate host animal, is typicallyimmunized with an immunizing agent to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro.

For anti-DNA98853 polypeptide antibodies, the immunizing agent willtypically include the DNA98853 polypeptide or a fusion protein thereof.For anti-DNA101848 polypeptide antibodies, the immunizing agent willtypically include the DNA101848 polypeptide or a fusion protein thereof.Generally, either peripheral blood lymphocytes (“PBLs”) are used ifcells of human origin are desired, or spleen cells or lymph node cellsare used if non-human mammalian sources are desired. The lymphocytes arethen fused with an immortalized cell line using a suitable fusing agent,such as polyethylene glycol, to form a hybridoma cell [Goding,Monoclonal Antibodies: Principles and Practice, Academic Press, (1986)pp. 59-103]. Immortalized cell lines are usually transformed mammaliancells, particularly myeloma cells of rodent, bovine and human origin.Usually, rat or mouse myeloma cell lines are employed. The hybridomacells may be cultured in a suitable culture medium that preferablycontains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lackthe enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT orHPRT), the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (“HAT medium”), whichsubstances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against aDNA98853 polypeptide or a DNA101848 polypeptide. Preferably, the bindingspecificity of monoclonal antibodies produced by the hybridoma cells isdetermined by immunoprecipitation or by an in vitro binding assay, suchas radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay(ELISA). Such techniques and assays are known in the art. The bindingaffinity of the monoclonal antibody can, for example, be determined bythe Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220(1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

3. Humanized Antibodies

The anti-DNA98853 polypeptide antibodies and anti-DNA101848 polypeptideantibodies of the invention may further comprise humanized antibodies orhuman antibodies. Humanized forms of non-human (e.g., murine) antibodiesare chimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin [Jones et al., Nature, 321:522-525(1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol. 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)].

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is for aDNA98853 polypeptide or for a DNA101848 polypeptide, and the other oneis for any other antigen, preferably for a cell-surface protein orreceptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J. 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

G. Uses for anti-DNA98853 Polypeptide Antibodies and for anti-DNA101848Polypeptide Antibodies

The anti-DNA98853 polypeptide antibodies and anti-DNA101848 polypeptideantibodies of the present invention have various utilities. Theanti-DNA98853 polypeptide antibodies or anti-DNA101848 polypeptideantibodies may be used in therapy, using techniques and methods ofadministration described above. Also, for example, anti-DNA98853polypeptide antibodies and anti-DNA101848 polypeptide antibodies may beused in diagnostic assays for the corresponding polypeptides, e.g.,detecting expression in specific cells, tissues, or serum. Variousdiagnostic assay techniques known in the art may be used, such ascompetitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Anti-DNA98853 polypeptide antibodies also are useful for the affinitypurification of DNA98853 polypeptides from recombinant cell culture ornatural sources. In this process, the antibodies against a DNA98853polypeptide are immobilized on a suitable support, such a Sephadex resinor filter paper, using methods well known in the art. The immobilizedantibody then is contacted with a sample containing the DNA98853polypeptide to be purified, and thereafter the support is washed with asuitable solvent that will remove substantially all the material in thesample except the DNA98853 polypeptide, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent that will release the DNA98853 polypeptide from theantibody.

Anti-DNA101848 polypeptide antibodies also are useful for the affinitypurification of DNA101848 polypeptides from recombinant cell culture ornatural sources. In this process, the antibodies against a DNA101848polypeptide are immobilized on a suitable support, such a Sephadex resinor filter paper, using methods well known in the art. The immobilizedantibody then is contacted with a sample containing the DNA101848polypeptide to be purified, and thereafter the support is washed with asuitable solvent that will remove substantially all the material in thesample except the DNA101848 polypeptide, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent that will release the DNA101848 polypeptide from theantibody.

H. Articles of Manufacture

An article of manufacture such as a kit containing DNA98853 polypeptide,DNA101848 polypeptide, or antibodies thereto useful for the diagnosis ortreatment of the disorders described herein comprises at least acontainer and a label. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition that is effective for diagnosing or treating thecondition and may have a sterile access port (for example, the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). The active agent in the compositionis the DNA98853 polypeptide, the DNA101848 polypeptide, or an antibodythereto. The label on, or associated with, the container indicates thatthe composition is used for diagnosing or treating the condition ofchoice. The article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution, and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use. The article ofmanufacture may also comprise a second or third container with anotheractive agent as described above.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1 Isolation of cDNA Clones

Encoding Human DNA98853 Polypeptide

Based upon the DNA sequence of Incyte clone 509 1511H (SEQ ID NO:7)shown in FIG. 9 (from the Incyte Pharmaceuticals LIFESEQ™ database),oligonucleotides were synthesized to identify by PCR a cDNA library thatcontained the sequence of interest. These oligonucleotides were:

Forward primer: (SEQ ID NO:8) 5′ GAGGGGGCTGGGTGAGATGTG 3′ (509-1)Reverse primer: (SEQ ID NO:9) 5′ TGCTTTTGTACCTGCGAGGAGG 3′ (509-4AS)

To isolate the full length coding sequence for DNA98853 polypeptide, aninverse long distance PCR procedure was carried out (FIG. 6). The PCRprimers generally ranged from 20 to 30 nucleotides. For inverse longdistance PCR, primer pairs were designed in such a way that the 5′ to 3′direction of each primer pointed away from each other.

A pair of inverse long distance PCR primers for cloning DNA98853 weresynthesized:

Primer 1 (left primer): (SEQ ID NO:10) 5′ pCATGGTGGGAAGGCCGGTAACG3′ (509-P5) Primer 2 (right primer): (SEQ ID NO:11)5′ pGATTGCCAAGAAAATGAGTACTGGGACC 3′ (509-P6)

In the inverse long distance PCR reaction, the template is plasmid cDNAlibrary. As a result, the PCR products contain the entire vectorsequence in the middle with insert sequences of interest at both ends.After the PCR reaction, the PCR mixture was treated with Dpn I whichdigests only the template plasmids, followed by agarose gel purificationof PCR products of larger than the size of the library cloning vector.Since the primers used in the inverse long distance PCR were also5′-phosphorylated, the purified products were then self-ligated andtransformed into E. coli competent cells. Colonies were screened by PCRusing 5′ vector primer and proper gene specific primer to identifyclones with larger 5′ sequence. Plasmids prepared from positive cloneswere sequenced. If necessary, the process could be repeated to obtainmore 5′ sequences based on new sequence obtained from the previousround.

The purpose of inverse long distance PCR is to obtain the completesequence of the gene of interest. The clone containing the full lengthcoding region was then obtained by conventional PCR.

The primer pair used to clone the full length coding region of DNA98853were synthesized:

Forward primer: (SEQ ID NO:12) 5′ ggaggatcgatACCATGGATTGCCAAGAAAATGAG 3′(Cla-MD-509) Reverse primer: (SEQ ID NO:13)5′ ggaggagcggccgcttaAGGGCTGGGAACTTCAAAGGGCAC (509.TAA.not)

For cloning purposes, a Cla I site and a Not I site were included in theforward primer and reverse primer respectively.

To ensure the accuracy of the PCR products, independent PCR reactionswere performed and several cloned products were sequenced.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for DNA98853 polypeptide (herein designated asDNA98853-1739) and the derived protein sequence for DNA98853polypeptide.

The entire nucleotide sequence of DNA98853 is shown in FIG. 1 (SEQ IDNO:1). Clone DNA98853-1739 has been deposited with ATCC and is assignedATCC Deposit No. ATCC 203906. Clone DNA98853 contains a single openreading frame with an apparent translational initiation site atnucleotide positions 4-6 and ending at the stop codon at nucleotidepositions 901-903 (FIG. 1). The predicted polypeptide precursor is 299amino acids long (FIG. 2). The full-length DNA98853 polypeptide proteinshown in FIG. 2 has an estimated molecular weight of about 3.3kilodaltons and a pI of about 4.72. A potential N-glycosylation siteexists between amino acids 74 and 77 of the amino acid sequence shown inFIG. 2. A potential N-myristoylation site exists between amino acids 24and 29 of the amino acid sequence shown in FIG. 2. Potential caseinkinase II phosphorylation sites exist between amino acids 123-126,185-188, 200-203, 252-255, 257-260, 271-274, and 283-286 of the aminoacid sequence shown in FIG. 2. A potential transmembrane domain existsbetween amino acids 137 to 158 of the sequence shown in FIG. 2. It ispresently believed that the polypeptide does not include a signalsequence.

Analysis of the amino acid sequence of the full-length DNA98853polypeptide suggests that portions of it possess homology to members ofthe tumor necrosis factor receptor family, thereby indicating thatDNA98853 polypeptide may be a novel member of the tumor necrosis factorreceptor family. There are three apparent extracellular cysteine-richdomains characteristic of the TNFR family [see, Naismith and Sprang,Trends Biochem. Sci., 23:74-79 (1998)], of which the first two CRDs have6 cysteines while the third CRD has 4 cysteines.

Example 2 Isolation of cDNA Clones Encoding Human DNA101848 Polypeptide

Based upon the DNA sequence of Incyte clone 509 1511H shown in FIG. 9(SEQ ID NO:7), oligonucleotides were synthesized to identify by PCR acDNA library that contained the sequence of interest. Theseoligonucleotides were:

Forward primer: (SEQ ID NO:8) 5′ GAGGGGGCTGGGTGAGATGTG 3′ (509-1)Reverse primer: (SEQ ID NO:9) 5′ TGCTTTTGTACCTGCGAGGAGG 3′ (509-4AS).

To isolate the full length coding sequence for DNA101848 polypeptide, aninverse long distance PCR procedure was carried out (FIG. 6). The PCRprimers generally ranged from 20 to 30 nucleotides. For inverse longdistance PCR, primer pairs were designed in such a way that the 5′ to 3′direction of each primer pointed away from each other.

A pair of inverse long distance PCR primers for cloning DNA101848 weresynthesized:

Primer 1 (left primer): (SEQ ID NO:10) 5′ pCATGGTGGGAAGGCCGGTAACG3′ (509-P5) Primer 2 (right primer): (SEQ ID NO:11)5′ pGATTGCCAAGAAAATGAGTACTGGGACC 3′ (509-P6)

In the inverse long distance PCR reaction, the template is plasmid cDNAlibrary. As a result, the PCR products contain the entire vectorsequence in the middle with insert sequences of interest at both ends.After the PCR reaction, the PCR mixture was treated with Dpn I whichdigests only the template plasmids, followed by agarose gel purificationof PCR products of larger than the size of the library cloning vector.Since the primers used in the inverse long distance PCR were also5′-phosphorylated, the purified products were then self-ligated andtransformed into E. coli competent cells. Colonies were screened by PCRusing 5′ vector primer and proper gene specific primer to identifyclones with larger 5′ sequence. Plasmids prepared from positive cloneswere sequenced. If necessary, the process could be repeated to obtainmore 5′ sequences based on new sequence obtained from the previousround.

The primer pair used to clone the full length coding region of DNA101848were synthesized:

Forward primer: (SEQ ID NO:12) 5′ ggaggatcgatACCATGGATTGCCAAGAAAATGAG 3′(Cla-MD-509) Reverse primer: (SEQ ID NO:13)5′ ggaggagcggccgcttaAGGGCTGGGAACTTCAAAGGGCAC (509.TAA.not)

For cloning purposes, a Cla I site and a Not I site were included in theforward primer and reverse primer respectively.

To ensure the accuracy of the PCR products, independent PCR reactionswere performed and several cloned products were sequenced.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for DNA101848 polypeptide (herein designated asDNA101848-1739) and the derived protein sequence for DNA101848polypeptide.

The entire nucleotide sequence of DNA101848 is shown in FIG. 3 (SEQ IDNO:4). Clone DNA101848-1739 has been deposited with ATCC and is assignedATCC Deposit No. ATCC 203907. Clone DNA101848 contains a single openreading frame with an apparent translational initiation site atnucleotide positions 4-6 and ending at the stop codon at nucleotidepositions 895-897 (FIG. 3). The predicted polypeptide precursor is 297amino acids long (FIG. 4). The full-length DNA101848 polypeptide proteinshown in FIG. 4 has an estimated molecular weight of about 3.28kilodaltons and a pI of about 4.72. A potential N-glycosylation siteexists between amino acids 74 and 77 of the amino acid sequence shown inFIG. 4. A potential N-myristoylation site exists between amino acids 24and 29 of the amino acid sequence shown in FIG. 4. Potential caseinkinase II phosphorylation sites exist between amino acids 123-126,185-188, 200-203, 252-255, 257-260, 271-274, and 283-286 of the aminoacid sequence shown in FIG. 4. A potential transmembrane domain existsbetween amino acids 137 to 158 of the sequence shown in FIG. 4. It ispresently believed that the polypeptide does not include a signalsequence.

Analysis of the amino acid sequence of the full-length DNA101848polypeptide suggests that portions of it possess homology to members ofthe tumor necrosis factor receptor family, thereby indicating thatDNA101848 polypeptide may be a novel member of the tumor necrosis factorreceptor family. There are three apparent extracellular cysteine-richdomains characteristic of the TNFR family [see, Naismith and Sprang,Trends Biochem. Sci. 23:74-79 (1998)], of which the first two CRDs have6 cysteines while the third CRD has 4 cysteines.

To further demonstrate that DNA101848 is indeed a transmembrane protein,two versions of epitope-tagged expression plasmids of DNA101848 wereconstructed in pRK5B (see Example 11), one with an N-terminal Flag-tag(Flag-DNA101848) and the other with a C-terminal Flag-tag(DNA101848-Flag). MCF-7 cells (ATCC) transfected with either construct(using Lipofectamine reagent; Gibco-BRL) were immunostained with M2anti-Flag antibody (Sigma) either with or without permeabilization with0.5% Triton X-100 in PBS. Cell staining was visualized by subsequentincubation with Cy₃-conjugated goat anti-mouse (Sigma). As shown FIG.10, without membrane permeabilization (FIGS. 10A and 10B), cell surfacestaining by M2 antibody was only seen in cells transfected withFlag-DNA101848 but not DNA101848-Flag. When cells were permeabilizedbefore anti-Flag immunostaining, comparable expressions were observedfor both types of constructs (FIGS. 10C and 10D). This experimentclearly demonstrated that DNA101848 is expressed as a cell surfaceprotein with N-terminal region outside of the cells and C-terminusregion inside of the cells. Therefore, DNA101848 represents a type IIItransmembrane protein.

Example 3 Use of DNA98853 Polypeptide-Encoding DNA or DNA101848Polypeptide-Encoding DNA as a Hybridization Probe

The following method describes use of a nucleotide sequence encodingDNA98853 polypeptide or a nucleotide sequence encoding DNA101848polypeptide as a hybridization probe.

DNA comprising the coding sequence of full-length DNA98853 polypeptide(as shown in FIG. 1, SEQ ID NO: 1) or a fragment thereof is employed asa probe to screen for homologous DNAs (such as those encodingnaturally-occurring variants of DNA98853 polypeptide) in human tissuecDNA libraries or human tissue genomic libraries. Similarly, DNAcomprising the coding sequence of full-length DNA101848 polypeptide (asshown in FIG. 3, SEQ ID NO:4) or a fragment thereof is employed as aprobe to screen for homologous DNAs (such as those encodingnaturally-occurring variants of DNA101848 polypeptide) in human tissuecDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled DNA98853 polypeptide-derived probe or of radiolabeledDNA101848 polypeptide-derived probe to the filters is performed in asolution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate,50 mM sodium phosphate, pH 6.8, 2×Denhardt's solution, and 10% dextransulfate at 42° C. for 20 hours. Washing of the filters is performed inan aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence DNA98853 polypeptide or with the DNAencoding full-length native sequence DNA101848 polypeptide can then beidentified using standard techniques known in the art.

Example 4 Expression of DNA98853 Polypeptides or DNA101848 Polypeptidesin E. coli

This example illustrates the preparation of forms of DNA98853polypeptides and forms of DNA101848 polypeptides by recombinantexpression in E. coli.

For expression of DNA98853 polypeptide, the DNA sequence encoding thefull-length DNA98853 polypeptide (SEQ ID NO:1) or a fragment or variantthereof is initially amplified using selected PCR primers. Forexpression of DNA101848 polypeptide, the DNA sequence encoding thefull-length DNA101848 polypeptide (SEQ ID NO:4) or a fragment or variantthereof is initially amplified using selected PCR primers.

The primers should contain restriction enzyme sites which correspond tothe restriction enzyme sites on the selected expression vector. Avariety of expression vectors may be employed. An example of a suitablevector is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95(1977)) which contains genes for ampicillin and tetracycline resistance.The vector is digested with restriction enzyme and dephosphorylated. ThePCR amplified sequences are then ligated into the vector. The vectorwill preferably include sequences which encode for an antibioticresistance gene, a trp promoter, a polyhis leader (including the firstsix STII codons, polyhis sequence, and enterokinase cleavage site), theDNA98853 polypeptide coding region or the DNA101848 polypeptide codingregion, lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized DNA98853 polypeptide or the solubilized DNA101848polypeptide can then be purified using a metal chelating column underconditions that allow tight binding of the polypeptide.

Example 5 Expression of DNA98853 Polypeptides or DNA101848 Polypeptidesin Mammalian Cells

This example illustrates preparation of forms of DNA98853 polypeptidesand DNA101848 polypeptides by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the DNA98853 polypeptide-encodingDNA is ligated into pRK5 with selected restriction enzymes to allowinsertion of the DNA98853 polypeptide-encoding DNA using ligationmethods such as described in Sambrook et al., supra. The resultingvector is called pRK5-DNA98853 polypeptide. Optionally, the DNA101848polypeptide-encoding DNA is ligated into pRK5 with selected restrictionenzymes to allow insertion of the DNA101848 polypeptide-encoding DNAusing ligation methods such as described in Sambrook et al., supra. Theresulting vector is called pRK5-DNA101848 polypeptide.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μgpRK5-DNA98853 polypeptide DNA is mixed with about 1 microgram DNAencoding the VA RNA gene [Thimmappaya et al., Cell 31:543 (1982)] anddissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂.Alternatively, about 10 microgram pRK5-DNA101848 polypeptide DNA ismixed with about 1μ DNA encoding the VA RNA gene [Thimmappaya et al.,Cell, 31:543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl, 0.1 mMEDTA, 0.227 M CaCl₂. To the vector mixture is added, dropwise, 500 μl of50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄, and a precipitate isallowed to form for 10 minutes at 25° C. The precipitate is suspendedand added to the 293 cells and allowed to settle for about four hours at37° C. The culture medium is aspirated off and 2 ml of 20% glycerol inPBS is added for 30 seconds. The 293 cells are then washed with serumfree medium, fresh medium is added and the cells are incubated for about5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of DNA98853 polypeptide or the presence of DNA101848polypeptide. The cultures containing transfected cells may undergofurther incubation (in serum free medium) and the medium is tested inselected bioassays.

In an alternative technique, DNA98853 polypeptide-encoding DNA orDNA101848 polypeptide-encoding DNA may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and followed by addition of 700microgram pRK5-DNA98853 polypeptide DNA, or by addition of 700 μgDNA101848 polypeptide DNA. The cells are first concentrated from thespinner flask by centrifugation and washed with PBS. The DNA-dextranprecipitate is incubated on the cell pellet for four hours. The cellsare treated with 20% glycerol for 90 seconds, washed with tissue culturemedium, and re-introduced into the spinner flask containing tissueculture medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine transferrin.After about four days, the conditioned media is centrifuged and filteredto remove cells and debris. The sample containing expressed DNA98853polypeptide or expressed DNA101848 polypeptide can then be concentratedand purified by any selected method, such as dialysis and/or columnchromatography.

In another embodiment, DNA98853 polypeptide or DNA101848 polypeptide canbe expressed in CHO cells. The pRK5-DNA98853 polypeptide vector or thepRK5-DNA101848 polypeptide vector can be transfected into CHO cellsusing known reagents such as CaPO₄ or DEAE-dextran. As described above,the cell cultures can be incubated, and the medium replaced with culturemedium (alone) or medium containing a radiolabel such as ³⁵S-methionine.After determining the presence of the desired polypeptide, the culturemedium may be replaced with serum free medium. Preferably, the culturesare incubated for about 6 days, and then the conditioned medium isharvested. The medium containing the expressed DNA98853 polypeptide orDNA101848 polypeptide can then be concentrated and purified by anyselected method.

Epitope-tagged DNA98853 polypeptide or epitope-tagged DNA101848polypeptide may also be expressed in host CHO cells. The DNA98853polypeptide-encoding DNA or the DNA101848 polypeptide-encoding DNA maybe subcloned out of the pRK5 vector. The subclone insert can undergo PCRto fuse in frame with a selected epitope tag such as a poly-his tag intoa Baculovirus expression vector. The poly-his tagged DNA98853polypeptide-encoding DNA insert or the poly-his tagged DNA101848polypeptide-encoding DNA insert can then be subcloned into an SV40driven vector containing a selection marker such as DHFR for selectionof stable clones. Finally, the CHO cells can be transfected (asdescribed above) with the SV40 driven vector. Labeling may be performed,as described above, to verify expression. The culture medium containingthe expressed poly-His tagged DNA98853 polypeptide or the expressedpoly-His tagged DNA101848 polypeptide can then be concentrated andpurified by any selected method, such as by Ni²⁺-chelate affinitychromatography.

Example 6 Expression of a DNA98853 Polypeptide or a DNA101848Polypeptide in Yeast

The following method describes recombinant expression of DNA98853polypeptides and DNA101848 polypeptides in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of DNA98853 polypeptide from the ADH2/GAPDHpromoter. DNA encoding the DNA98853 polypeptide of interest, a selectedsignal peptide and the promoter is inserted into suitable restrictionenzyme sites in the selected plasmid to direct intracellular expressionof the DNA98853 polypeptide. For secretion, DNA encoding the DNA98853polypeptide can be cloned into the selected plasmid, together with DNAencoding the ADH2/GAPDH promoter, the yeast alpha-factor secretorysignal/leader sequence, and linker sequences (if needed) for expressionof the DNA98853 polypeptide.

Alternatively, yeast expression vectors are constructed forintracellular production or secretion of DNA101848 polypeptide from theADH2/GAPDH promoter. DNA encoding the DNA101848 polypeptide of interest,a selected signal peptide and the promoter is inserted into suitablerestriction enzyme sites in the selected plasmid to direct intracellularexpression of the DNA101848 polypeptide. For secretion, DNA encoding theDNA101848 polypeptide can be cloned into the selected plasmid, togetherwith DNA encoding the ADH2/GAPDH promoter, the yeast alpha-factorsecretory signal/leader sequence, and linker sequences (if needed) forexpression of the DNA101848 polypeptide.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant DNA98853 polypeptide or DNA101848 polypeptide cansubsequently be isolated and purified by removing the yeast cells fromthe fermentation medium by centrifugation and then concentrating themedium using selected cartridge filters. The concentrate containing theDNA98853 polypeptide or DNA101848 polypeptide may further be purifiedusing selected column chromatography resins.

Example 7 Expression of DNA98853 Polypeptide or DNA101848 Polypeptidesin Baculovirus-Infected Insect Cells

The following method describes recombinant expression of DNA98853polypeptides and DNA101848 polypeptides in Baculovirus-infected insectcells.

The DNA98853 polypeptide-encoding DNA or the DNA101848polypeptide-encoding DNA is fused upstream of an epitope tag containedwithin a baculovirus expression vector. Such epitope tags includepoly-his tags and immunoglobulin tags (like Fc regions of IgG). Avariety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, theDNA98853 polypeptide-encoding DNA or the desired portion of the DNA98853polypeptide-encoding DNA (such as the sequence encoding theextracellular domain of a transmembrane protein) is amplified by PCRwith primers complementary to the 5′ and 3′ regions. Alternatively, theDNA101848 polypeptide-encoding DNA or the desired portion of theDNA101848 polypeptide-encoding DNA (such as the sequence encoding theextracellular domain of a transmembrane protein) is amplified by PCRwith primers complementary to the 5′ and 3′ regions. The 5′ primer mayincorporate flanking (selected) restriction enzyme sites. The product isthen digested with those selected restriction enzymes and subcloned intothe expression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4 to 5 days of incubation at 28° C.,the released viruses are harvested and used for further amplifications.Viral infection and protein expression is performed as described byO'Reilley et al., Baculovirus expression vectors: A laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged DNA98853 polypeptide or expressed poly-histagged DNA101848 polypeptide can then be purified, for example, byNi²⁺-chelate affinity chromatography as follows. Extracts are preparedfrom recombinant virus-infected Sf9 cells as described by Rupert et al.,Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspendedin sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA;10% Glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 secondson ice. The sonicates are cleared by centrifugation, and the supernatantis diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%Glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni²⁺-NTAagarose column (commercially available from Qiagen) is prepared with abed volume of 5 mL, washed with 25 mL of water and equilibrated with 25mL of loading buffer. The filtered cell extract is loaded onto thecolumn at 0.5 mL per minute. The column is washed to baseline A₂₈₀ withloading buffer, at which point fraction collection is started. Next, thecolumn is washed with a secondary wash buffer (50 mM phosphate; 300 mMNaCl, 10% Glycerol, pH 6.0), which elutes nonspecifically bound protein.After reaching A₂₈₀ baseline again, the column is developed with a 0 to500 mM Imidazole gradient in the secondary wash buffer. One mL fractionsare collected and analyzed by SDS-PAGE and silver staining or westernblot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen).Fractions containing the eluted His₁₀-tagged DNA98853 polypeptide or theeluted His₁₀-tagged DNA101848 polypeptide are pooled and dialyzedagainst loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) DNA98853polypeptide or the IgG tagged (or Fc tagged) DNA101848 polypeptide canbe performed using known chromatography techniques, including forinstance, Protein A or protein G column chromatography.

Example 8 Preparation of Antibodies that Bind DNA98853 Polypeptidesand/or DNA101848 Polypeptides

This example illustrates the preparation of monoclonal antibodies whichcan specifically bind to DNA98853 polypeptides and/or DNA101848polypeptides.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified DNA98853 polypeptide, purified DNA101848polypeptide, fusion proteins containing a DNA98853 polypeptide, fusionproteins containing a DNA101848 polypeptide, cells expressingrecombinant DNA98853 polypeptide on the cell surface, and cellsexpressing recombinant DNA101848 polypeptide on the cell surface.Selection of the immunogen can be made by the skilled artisan withoutundue experimentation.

Mice, such as Balb/c, are immunized with the DNA98853 polypeptideimmunogen, or DNA101848 polypeptide immunogen, emulsified in completeFreund's adjuvant and injected subcutaneously or intraperitoneally in anamount from 1-100 micrograms. Alternatively, the immunogen is emulsifiedin MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) andinjected into the animal's hind foot pads. The immunized mice are thenboosted 10 to 12 days later with additional immunogen emulsified in theselected adjuvant. Thereafter, for several weeks, the mice may also beboosted with additional immunization injections. Serum samples may beperiodically obtained from the mice by retro-orbital bleeding fortesting in ELISA assays to detect anti-DNA98853 polypeptide antibodiesor DNA101848 polypeptide antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of DNA98853 polypeptide or of DNA101848 polypeptide. Three tofour days later, the mice are sacrificed and the spleen cells areharvested. The spleen cells are then fused (using 35% polyethyleneglycol) to a selected murine myeloma cell line such as P3X63AgU.1,available from ATCC, No. CRL 1597. The fusions generate hybridoma cellswhich can then be plated in 96 well tissue culture plates containing HAT(hypoxanthine, aminopterin, and thymidine) medium to inhibitproliferation of non-fused cells, myeloma hybrids, and spleen cellhybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstDNA98853 polypeptide or for reactivity against DNA101848 polypeptide.Determination of “positive” hybridoma cells secreting the desiredmonoclonal antibodies against a DNA98853 polypeptide or a DNA101848polypeptide is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-DNA98853polypeptide monoclonal antibodies or anti-DNA101848 polypeptidemonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 9 Assays to Detect Expression of DNA101848 Polypeptide mRNA inHuman Cells and Tissues

Northern Blotting was conducted according to common procedures known tothose of skill in the art. Briefly, human polyA+ RNA normal tissue blotsor tumor cell line blots (Clontech) were hybridized according to themanufacturer's instructions. ³²P-labeled probes were generated using DNAfragments corresponding to the nucleotides 478-903 of DNA101848 (SEQ IDNO:4). As shown in FIG. 7, relatively high expression levels weredetected in two human tumor cell lines, lung carcinoma A549 and melanomaG361. Relatively weak expression levels were also found in prostate,testis, ovary, thyroid, spinal cord and adrenal gland tissues.Interestingly, a smaller transcript with relatively high expressionlevel existed in stomach.

Example 10 Activation of NF-KB

An assay was conducted to determine whether DNA98853 polypeptide orDNA101848 polypeptide induces NF-κB activation by analyzing expressionof a reporter gene driven by a promoter containing a NF-κB responsiveelement from the E-selectin gene.

Human 293 cells (2×10⁵) (maintained in HG-DMEM supplemented with 10%FBS, 2 mM glutamine, 100 microgram/ml penicillin, and 100 microgramstreptomycin) were transiently transfected by calcium phosphatetransfection with 0.5 microgram of the firefly luciferase reporterplasmid pGL3.ELAM.tk [Yang et al., Nature, 395:284-288 (1998)] and 0.05microgram of the Renilla luciferase reporter plasmid (as internaltransfection control) (Promega), as well as the indicated additionalexpression vectors for DNA98853 polypeptide or DNA101848 polypeptide(described above), and carrier plasmid pRK5D to maintain constant DNAbetween transfections. After 24 hours, the transfected cells wereharvested and luciferase activity was assayed as recommended by themanufacturer (Promega). Activities (average of triplicate wells) werenormalized for differences in transfection efficiency by dividingfirefly luciferase activity by that of Renilla luciferase activity andwere expressed as activity relative to that seen in the absence of addedexpression vectors.

As shown in FIG. 8A, overexpression of flag-tagged DNA101848 polypeptideresulted in significant reporter gene activation. Similar activity wasobtained for DNA98853 polypeptide (data not shown).

For the following experiments, only DNA101848 polypeptide was used.

To examine potential intracellular mediators of the DNA 101848polypeptide signaling, dominant negative mutants of certainintracellular signaling molecules involved in the pathways of NF-KBactivation by TNF-alpha, IL-1, or LPs-Toll were tested.

The 293 cells were transiently transfected (as above) with thepGL3.ELAM.tk and expression vectors. In addition, the cells weretransfected with the following mammalian expression vectors encodingdominant negative forms of TRAF2-DN (aa 87-501); TRAF6-DN (aa 289-522);and NIK-DN [described in Cao et al., Science 271:1128-1131 (1996);Malinin et al., Nature, 385:540-544 (1997); Muzio et al., Science,278:1362-1365 (1997); Rothe et al., Science, 269:1424-1427 (1995); Tinget al., EMBO J. 15:6189-6196 (1996); Wesche et al., Immunity, 7:837-847(1997)]. Luciferase activity was expressed and determined as describedabove.

The results are shown in FIG. 8B. Co-transfection of a kinase-inactivemutant form of NIK, which acts as a dominant inhibitor of NF-KBactivation by TNF-alpha (Malinin et al., Nature, 385:540-544 (1997)),IL-1 (Malinin et al., supra), and LPs-Toll (Yang et al., Nature,395:284-288 (1998)), substantially blocked NF-KB activation throughDNA101848 polypeptide. A dominant negative TRAF2 or dominant negativeTraf-6 (Rothe et al., Science, 269:1424-1427 (1995); Rothe et al., Cell:78:681-692 (1994)) possessing an N-terminal deletion also attenuatedNF-KB activation (FIG. 8C). Accordingly, it appears that DNA101848polypeptide activates NF-KB predominantly through TRAF-2 and TRAF-6.

Example 11 Identification of a Ligand for the DNA101848 Receptor

A chimeric molecule, referred to herein as “AP-EDA-A2”, was preparedusing human placenta alkaline phosphatase (AP) fused to the N-terminusof an EDA-A2 polypeptide consisting of amino acids 241-389 (Bayes etal., supra). The AP was obtained by PCR amplification using pAPtag-5(Genehunter Corporation) as a template, and fused and cloned into theexpression vector, pCMV-1 Flag (Sigma), with AP at the N-terminus ofEDA-A2. The AP-EDA-A2 was transiently transfected (using Lipofectaminereagent; Gibco-BRL) and expressed in human embryonic kidney 293 cells(ATCC). AP-TNF-alpha (Pennica et al., infra) and AP-TALL-1 (amino acids136-285; sequence disclosed in WO98/18921 published May 7, 1998; Mooreet al., Science, 285:260-263 (1999)) were similarly prepared. Theconditioned medium from the transfected 293 cells was filtered (0.45micron), stored at 4° C. in a buffer containing 20 mM Hepes (pH 7.0) and1 mM sodium azide, and used for subsequent cell staining procedures. Inaddition, a N-terminal Flag-tagged form of EDA-A2 was constructed in apCMV-1 Flag vector. To promote the trimerization of this Flag-taggedEDA-A2 construct, a trimeric form of leucine-zipper sequence [Science,262:1401-1407 (1993)] was inserted between the Flag-tag and the EDA-A2(consisting of amino acids 179-389; Bayes et al., supra), and thisconstruct was referred to as Flag-LZP-EDA-A2. Another form of Flagtagged EDA-A2 was also made by cloning amino acids 179-389 of EDA-A2into pCMV-1Flag vector, and referred to as Flag-EDA-A2. TheFlag-LZP-EDA-A2 or Flag-EDA-A2 was purified using M2-agarose gel (Sigma)from serum-free medium of 293 cells transfected with the correspondingexpressing plasmid. Flag-TALL-1 (consisting of amino acids 136-285;sequence disclosed in WO98/18921 published May 7, 1998; Moore et al.,Science, 285:260-263 (1999)) was generated in a similar way.

To identify a potential ligand for DNA101848 receptor, COS 7 cells(ATCC) were transiently transfected (using Lipofectamine reagent) withmembrane forms of various ligands of TNF family. Among the ligandstested were APRIL, TALL-1, 4-1 BBL, CD27L, CD30L, CD40L, EDA-A2, RANKL,TNF-alpha, and Apo2L/TRAIL.

Human TNF-alpha was cloned into pRK5B vector (pRK5B is a precursor ofpRK5D that does not contain the SfiI site; see Holmes et al., Science,253:1278-1280 (1991)). For the detection of TNF-alpha expression on thecell surface, a Flag tag was inserted between amino acid 70 and aminoacid 71 (using the numbering according to the sequence in Pennica etal., Nature, 312:724-729 (1984)). An extracellular region of TALL-1 (aa75-285; sequence disclosed in WO98/18921 published May 7, 1998; Moore etal., Science, 285:260-263 (1999)), 4-1BBL (aa 59-254; Goodwin et al.,Eur. J. Immunol., 23:2631-2641 (1993)), CD27 ligand (aa 40-193; Goodwinet al., Cell 73:447-456 (1993)), CD30 ligand (aa 61-234; Smith et al.,Cell, 73:1349-1360 (1993)), RANKL (aa 71-317; see WO98/28426), Apo-2ligand (aa 40-281; see WO97/25428) or Apo-3L (aa 46-249; see WO99/19490)was individually cloned at the BamHI site. This resulted in a chimericligand with the intracellular and transmembrane regions from TNF-alphaand the extracellular region from the various ligands. For APRIL (Hahneet al., J. Exp. Med., 188:1185-1190 (1998)) and EDA-A2 (Bayes et al.,supra), full length cDNA clones without Flag tag were used.

COS 7 (ATCC) cells transfected with various ligands were incubated withDNA101848-ECD-hFc or TNFR1-hFc (constructs described below) at 1 μg/mlfor 1 hour in PBS containing 5% goat serum (Sigma). Cells weresubsequently washed three times with PBS and fixed with 4%paraformaldehyde in PBS. Cell staining was visualized by incubation withbiotinylated goat anti-human antibody (Jackson Labs, at 1:200 dilution)followed by Cy3-streptavidin (Jackson Labs, at 1:200 dilution). Amongall the ligands tested, DNA101848-ECD-hFc only bound EDA-A2 transfectedcells. As shown in FIG. 11, DNA101848-hFc but not TNFR-hFc bound tocells transfected with EDA-A2.

To demonstrate the binding of soluble EDA-A2 to cell membrane bound formof DNA101848, COS 7 cells were transfected with 1 microgram DNA101848(cloned in pRK5B vector) or empty vector plasmid (pRK5B). 18-24 hoursafter transfection, cells were incubated with conditioned mediumcontaining AP-EDA-A2; AP-TNF-alpha; or AP-TALL-1 for 1 hour at roomtemperature and stained for AP activity in situ as described inTartaglia et al., Cell, 83:1263-1271 (1995). As shown in FIG. 12,AP-EDA-A2 but not AP-TNF-alpha or AP-TALL-1 specifically bound to cellstransfected with DNA101848.

To demonstrate the binding of soluble EDA-A2 to DNA101848 ECD-hFC, oneμg of the purified Flag-LZP-EDA-A2 or Flag-EDA-A2 was incubated with 1μg of purified human immunoadhesin containing the IgG1-Fc fusion of theECD of DNA101848 (DNA101848-ECD-hFc) or TNFR-hFC overnight at 4° C. induplicate. The DNA101848-ECD-hFc immunoadhesins were prepared by methodsdescribed in Ashkenazi et al., Proc. Natl. Acad. Sci., 88:10535-10539(1991). The immunoadhesin constructs consisted of amino acids 2-154 ofthe human DNA 101848 polypeptide (see FIG. 4). The DNA101848-ECDconstructs were expressed in CHO cells using a heterologous signalsequence (pre-pro trypsin amino acids 1-17 of pCMV-1 Flag (Sigma)) andencoding the human IgG1 Fc region downstream of the DNA101848 sequence,and then purified by protein A affinity chromatography. TNFR1-hFcconstruct was prepared as described in Ashkenazi et al., Proc. Natl.Acad. Sci., 88:10535-10539 (1991)). Human TNFRSF19-hFc containing aminoacids 1-169 (Hu et al., Genomics, 62:103-107 (1999)) was prepared as forTNFR1-hFc. The ligand-receptor complex was subjected toimmunoprecipitation through the receptor-immunoadhesin with proteinA-agarose (Repligen). The immunoprecipitates were then analyzed byWestern blot using anti-Flag M2 mAb (Sigma).

The data shows that Flag-LZP-EDA-A2 or Flag-EDA-A2 bound toDNA101848-hFC, but not to TNFR1-hFc or TNFRSF19-hFC (FIG. 13).

Example 12 Interaction Between DNA101848 with EDA-A2 Results inActivation of NF-kB

293 cells (ATCC) were seeded 24 hours before transfection at 1×10⁵cells/well into 12-well plates and transfected with 0.25 μg ofELAM-luciferase reporter gene plasmid, 25 ng pRL-TK (Promega) and theindicated amounts of each expression construct (see FIG. 14). Totalamount of transfected DNA was kept constant at 1 ug by supplementationwith empty pRK5B vector (see Example 11). In some assay wells,Flag-tagged ligands (prepared as described in Example 11) were added atconcentrations indicated 4 hours after transfection. In other assaywells, the cells were co-transfected with full length EDA-A2 (Bayes etal., supra) or TALL-1 (sequence disclosed in WO98/18921 published May 7,1998; Moore et al., Science, 285:260-263 (1999)). Cells were harvested20-24 hours after transfection and reporter gene activity determinedwith the Dual-Luciferase Reporter Assay System (Promega).

Only minimal activation of NF-kB was observed when DNA101848 wasexpressed alone at low levels (such as at 0.1 ng). The activation ofNF-kB, however, was greatly augmented by either addition of Flag-EDA-A2or by co-transfection with full length EDA-A2 (FIG. 14).

Treatment of untransfected 293E (Invitrogen) cells with Flag-EDA-A2 (0.2μg/ml) also resulted in activation of the NF-kB pathway (see FIG. 15A).This was measured by Western Blotting using anti-phospho-IKB-a (NewEngland BioLabs). Preincubation with 20 μg/ml DNA101848-ECD-hFc (seeExample 11) abolished IKB-a phosphorylation induced by Flag-EDA-A2 (FIG.15B). These results suggest that one physiological consequence ofDNA101848 and EDA-A2 interaction is activation of the NF-kB pathway.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. USA (ATCC):

Material ATCC Dep. No. Deposit Date DNA98853-1739 203906 Apr. 6, 1999DNA101848-1739 203907 Apr. 6, 1999

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit forthirty (30) years from the date of deposit and at least five years fromthe most recent request for the furnishing of a sample of the depositwas received from the depository. The deposits will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC Section 122 and the Commissioner's rulespursuant thereto (including 37 CFR Section 1.14 with particularreference to 886 OG 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. An isolated nucleic acid comprising DNA having at least 95% sequenceidentity to (a) a DNA molecule encoding a DNA98853 polypeptidecomprising the sequence of amino acid residues 1 to 299 of SEQ ID NO:3,or (b) the complement of the DNA molecule of (a).
 2. The nucleic acid ofclaim 1, wherein said DNA comprises the nucleotide sequence of SEQ IDNO:1 or its complement.
 3. The nucleic acid of claim 1, wherein said DNAcomprises nucleotides 1-900 of the nucleotide sequence of SEQ ID NO:1.4. An isolated nucleic acid comprising DNA having at least 95% sequenceidentity to (a) a DNA molecule encoding the same mature polypeptideencoded by the cDNA in ATCC Deposit No. 203906 (DNA98853), or (b) thecomplement of the DNA molecule of (a).
 5. The nucleic acid of claim 4which comprises a DNA molecule encoding the same mature polypeptideencoded by the cDNA in ATCC Deposit No. 203906 (DNA98853).
 6. Anisolated nucleic acid comprising DNA having at least 95% sequenceidentity to (a) a DNA molecule encoding a DNA98853 polypeptidecomprising the sequence of amino acid residues 1 to X of FIG. 2 (SEQ IDNO:3), or (b) the complement of the DNA molecule of (a), wherein X isany one of amino acid residues 131-141 of FIG. 2 (SEQ ID NO:3).
 7. Anisolated nucleic acid comprising DNA from the group consisting of: a) aDNA having at least 80% sequence identity to a DNA sequence encoding aDNA98853 polypeptide comprising amino acid residues 1 to 299 of FIG. 2(SEQ ID NO:3); b) a DNA sequence that hybridizes under stringentconditions to a DNA of a); c) a DNA sequence that, due to the degeneracyof the genetic code, encodes a DNA98853 polypeptide of a); and d) DNAcomplementary to the DNA of a), b), or c).
 8. A vector comprising thenucleic acid of claim
 1. 9. The vector of claim 8 operably linked tocontrol sequences recognized by a host cell transformed with the vector.10. A host cell comprising the vector of claim
 8. 11. The host cell ofclaim 10, wherein said cell is a CHO cell.
 12. The host cell of claim10, wherein said cell is an E. coli.
 13. The host cell of claim 10,wherein said cell is a yeast cell.
 14. A process for producing aDNA98853 polypeptide comprising culturing the host cell of claim 10under conditions suitable for expression of said DNA98853 polypeptideand recovering said DNA98853 polypeptide from the cell culture.
 15. Anisolated DNA98853 polypeptide comprising amino acid residues 1 to 299 ofFIG. 2 (SEQ ID NO:3).
 16. An isolated DNA98853 polypeptide encoded bythe cDNA insert of the vector deposited as ATCC Accession No. 203906(DNA98853).
 17. An isolated DNA98853 polypeptide comprising amino acidresidues 1 to X of FIG. 2 (SEQ ID NO:3), wherein X is any one of aminoacid residues 131-141 FIG. 2 (SEQ ID NO:3).
 18. An isolated DNA98853polypeptide comprising a polypeptide selected from the group consistingof: a) a DNA98853 polypeptide comprising amino acid residues 1 to X ofFIG. 2 (SEQ ID NO:3), wherein X is any one of amino acid residues131-141 of FIG. 2 (SEQ ID NO:3); and b) a fragment of a), wherein saidfragment is a biologically active polypeptide.
 19. A chimeric moleculecomprising a DNA98853 polypeptide fused to a heterologous amino acidsequence.
 20. The chimeric molecule of claim 19, wherein saidheterologous amino acid sequence is an epitope tag sequence.
 21. Thechimeric molecule of claim 19, wherein said heterologous amino acidsequence is a Fc region of an immunoglobulin.
 22. An antibody whichspecifically binds to a DNA98853 polypeptide.
 23. The antibody of claim22, wherein said antibody is a monoclonal antibody.
 24. A compositioncomprising an isolated DNA98853 polypeptide of claim 15 and a carrier.25. The composition of claim 24 wherein said carrier is apharmaceutically-acceptable carrier.
 26. An isolated nucleic acidcomprising DNA having at least 95% sequence identity to (a) a DNAmolecule encoding a DNA101848 polypeptide comprising the sequence ofamino acid residues 1 to 297 of FIG. 4 (SEQ ID NO:6), or (b) thecomplement of the DNA molecule of (a).
 27. The nucleic acid of claim 26,wherein said DNA comprises the nucleotide sequence of SEQ ID NO:4 or itscomplement.
 28. The nucleic acid of claim 26, wherein said DNA comprisesnucleotides 1-894 of the nucleotide sequence of SEQ ID NO:4.
 29. Anisolated nucleic acid comprising DNA having at least 95% sequenceidentity to (a) a DNA molecule encoding the same mature polypeptideencoded by the cDNA in ATCC Deposit No. 203907 (DNA101848), or (b) thecomplement of the DNA molecule of (a).
 30. The nucleic acid of claim 29which comprises a DNA molecule encoding the same mature polypeptideencoded by the cDNA in ATCC Deposit No. 203907 (DNA101848).
 31. Anisolated nucleic acid comprising DNA having at least 95% sequenceidentity to (a) a DNA molecule encoding a DNA101848 polypeptidecomprising the sequence of amino acid residues 1 to X of FIG. 4 (SEQ IDNO:6), or (b) the complement of the DNA molecule of (a), wherein X isany one of amino acid residues 131-141 of FIG. 4 (SEQ ID NO:6).
 32. Anisolated nucleic acid comprising DNA from the group consisting of: a) aDNA having at least 80% sequence identity to a DNA sequence encoding aDNA101848 polypeptide comprising amino acid residues 1 to 297 of FIG. 4(SEQ ID NO:6); b) a DNA sequence that hybridizes under stringentconditions to a DNA of a); c) a DNA sequence that, due to the degeneracyof the genetic code, encodes a DNA101848 polypeptide of a); and d) DNAcomplementary to the DNA of a), b), or c).
 33. A vector comprising thenucleic acid of claim
 26. 34. The vector of claim 33 operably linked tocontrol sequences recognized by a host cell transformed with the vector.35. A host cell comprising the vector of claim
 33. 36. The host cell ofclaim 35, wherein said cell is a CHO cell.
 37. The host cell of claim35, wherein said cell is an E. coli.
 38. The host cell of claim 35,wherein said cell is a yeast cell.
 39. A process for producing aDNA101848 polypeptide comprising culturing the host cell of claim 35under conditions suitable for expression of said DNA101848 polypeptideand recovering said DNA101848 polypeptide from the cell culture.
 40. Anisolated DNA101848 polypeptide comprising amino acid residues 1 to 297of FIG. 4 (SEQ ID NO:6).
 41. An isolated DNA101848 polypeptide encodedby the cDNA insert of the vector deposited as ATCC Accession No. 203907(DNA101848).
 42. An isolated DNA101848 polypeptide comprising amino acidresidues 1 to X of FIG. 4 (SEQ ID NO:6), wherein X is any one of aminoacid residues 131-141 of FIG. 4 (SEQ ID NO:6).
 43. An isolated DNA101848polypeptide comprising a polypeptide selected from the group consistingof: a) a DNA101848 polypeptide comprising amino acid residues 1 to X ofFIG. 4 (SEQ ID NO:6), wherein X is any one of amino acid residues131-141 of FIG. 4 (SEQ ID NO:6); and b) a fragment of a), wherein saidfragment is a biologically active polypeptide.
 44. A chimeric moleculecomprising a DNA101848 polypeptide fused to a heterologous amino acidsequence.
 45. The chimeric molecule of claim 44, wherein saidheterologous amino acid sequence is an epitope tag sequence.
 46. Thechimeric molecule of claim 44, wherein said heterologous amino acidsequence is a Fc region of an immunoglobulin.
 47. An antibody whichspecifically binds to a DNA101848 polypeptide.
 48. The antibody of claim47, wherein said antibody is a monoclonal antibody.
 49. A compositioncomprising an isolated DNA101848 polypeptide of claim 40 and a carrier.50. The composition of claim 49 wherein said carrier is apharmaceutically-acceptable carrier.
 51. A method of modulatingapoptosis in mammalian cells, comprising exposing said cells to aneffective amount of DNA98853 polypeptide or DNA101848 polypeptide.
 52. Amethod of modulating NF-KB activation in mammalian cells, comprisingexposing said cells to an effective amount of DNA98853 polypeptide orDNA101848 polypeptide.
 53. A method of modulating a proinflammatory orautoimmune response in mammalian cells, comprising exposing said cellsto an effective amount of DNA98853 polypeptide or DNA101848 polypeptide.54. A method of inhibiting or neutralizing EDA-A2 polypeptide biologicalactivity in mammalian cells, comprising exposing said mammalian cells toan effective amount of EDA-A2 polypeptide antagonist.
 55. The method ofclaim 54 wherein said EDA-A2 polypeptide antagonist comprises aDNA101848 or DNA98853 receptor immunoadhesin.
 56. The method of claim 55wherein said DNA101848 receptor immunoadhesin comprises a DNA101848extracellular domain sequence fused to an Fc region of animmunoglobulin.
 57. The method of claim 54 wherein said EDA-A2polypeptide antagonist comprises a DNA101848 receptor linked to anonproteinaceous polymer selected from the group consisting ofpolyethylene glycol, polypropylene glycol, and polyoxyalkylene.
 58. Themethod of claim 54 wherein said EDA-A2 polypeptide antagonist comprisesa DNA101848 receptor polypeptide or DNA98853 receptor polypeptide linkedto a heterologous sequence.
 59. The method of claim 58 wherein saidheterologous sequence is an epitope tag sequence.
 60. The method ofclaim 58 wherein said heterologous sequence is a leucine zippersequence.
 61. The method of claim 54 wherein said EDA-A2 polypeptideantagonist comprises an antibody selected from the group consisting of aDNA101848 receptor antibody and a DNA98853 receptor antibody.